53 human secreted proteins

ABSTRACT

The present invention relates to novel human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human secreted proteins. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating diseases, disorders, and/or conditions related to these novel human secreted proteins.

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 60/244,591, filed Nov. 1, 2000; this application is also a continuation-in-part of, and claims priority under 35 U.S.C. § 120 to U.S. patent application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part, and claims priority under 35 U.S.C. § 120 to International Patent Application No: PCT/US98/21142, filed Oct. 8, 1998 (published in English), which claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos. 60/061,463, 60/061,529, 60/071,498, 60/061,527, 60/061,536, and 60/061,532, filed on Oct. 9, 1997. Each of the above referenced applications is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to novel proteins. More specifically, isolated nucleic acid molecules are provided encoding novel polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic methods useful for diagnosing, treating, preventing and/or prognosing disorders related to these novel polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further relates to methods and/or compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.

BACKGROUND OF THE INVENTION

[0003] Unlike bacterium, which exist as a single compartment surrounded by a membrane, human cells and other eukaryotes are subdivided by membranes into many functionally distinct compartments. Each membrane-bounded compartment, or organelle, contains different proteins essential for the function of the organelle. The cell uses “sorting signals,” which are amino acid motifs located within the protein, to target proteins to particular cellular organelles.

[0004] One type of sorting signal, called a signal sequence, a signal peptide, or a leader sequence, directs a class of proteins to an organelle called the endoplasmic reticulum (ER). The ER separates the membrane-bounded proteins from all other types of proteins. Once localized to the ER, both groups of proteins can be further directed to another organelle called the Golgi apparatus. Here, the Golgi distributes the proteins to vesicles, including secretory vesicles, the cell membrane, lysosomes, and the other organelles.

[0005] Proteins targeted to the ER by a signal sequence can be released into the extracellular space as a secreted protein. For example, vesicles containing secreted proteins can fuse with the cell membrane and release their contents into the extracellular space—a process called exocytosis. Exocytosis can occur constitutively or after receipt of a triggering signal. In the latter case, the proteins are stored in secretory vesicles (or secretory granules) until exocytosis is triggered. Similarly, proteins residing on the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of a “linker” holding the protein to the membrane.

[0006] Thus there exists a clear need for identifying and using novel secreted polynucleotides and polypeptides. Identification and sequencing of human genes is a major goal of modern scientific research. For example, by identifying genes and determining their sequences, scientists have been able to make large quantities of valuable human “gene products.” These include human insulin, interferon, Factor VIII, tumor necrosis factor, human growth hormone, tissue plasminogen activator, and numerous other compounds. Additionally, knowledge of gene sequences can provide the key to treatment or cure of genetic diseases (such as muscular dystrophy and cystic fibrosis).

SUMMARY OF THE INVENTION

[0007] The present invention relates to novel secreted proteins. More specifically, isolated nucleic acid molecules are provided encoding novel secreted polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic methods useful for diagnosing, treating, preventing and/or prognosing disorders related to these novel polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further relates to methods and/or compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.

DETAILED DESCRIPTION

[0008] Polynucleotides and Polypeptides

[0009] Description of Table 1A

[0010] Table 1A summarizes information concerning certain polynucleotides and polypeptides of the invention. The first column provides the gene number in the application for each clone identifier. The second column provides a unique clone identifier, “Clone ID”, for a cDNA clone related to each contig sequence disclosed in Table 1A. Third column, the cDNA Clones identified in the second column were deposited as indicated in the third column (i.e. by ATCC Deposit Number and deposit date). Some of the deposits contain multiple different clones corresponding to the same gene. In the fourth column, “Vector” refers to the type of vector contained in the corresponding cDNA Clone identified in the second column. In the fifth column, the nucleotide sequence identified as “NT SEQ ID NO: X” was assembled from partially homologous (“overlapping”) sequences obtained from the corresponding cDNA clone identified in the second column and, in some cases, from additional related cDNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO: X. In the sixth column, “Total NT Seq.” refers to the total number of nucleotides in the contig sequence identified as SEQ ID NO: X.” The deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as “5′ NT of Clone Seq.” (seventh column) and the “3′ NT of Clone Seq.” (eighth column) of SEQ ID NO: X. In the ninth column, the nucleotide position of SEQ ID NO: X of the putative start codon (methionine) is identified as “5′ NT of Start Codon.” Similarly, in column ten, the nucleotide position of SEQ ID NO: X of the predicted signal sequence is identified as “5′ NT of First AA of Signal Pep.” In the eleventh column, the translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO: Y,” although other reading frames can also be routinely translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.

[0011] In the twelfth and thirteenth columns of Table 1A, the first and last amino acid position of SEQ ID NO: Y of the predicted signal peptide is identified as “First AA of Sig Pep” and “Last AA of Sig Pep.” In the fourteenth column, the predicted first amino acid position of SEQ ID NO: Y of the secreted portion is identified as “Predicted First AA of Secreted Portion”. The amino acid position of SEQ ID NO: Y of the last amino acid encoded by the open reading frame is identified in the fifteenth column as “Last AA of ORF”.

[0012] SEQ ID NO: X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO: Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, SEQ ID NO: X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO: X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO: Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the secreted proteins encoded by the cDNA clones identified in Table 1A and/or elsewhere herein

[0013] Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).

[0014] Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO: X, and the predicted translated amino acid sequence identified as SEQ ID NO: Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCC, as set forth in Table 1A. The nucleotide sequence of each deposited plasrnid can readily be determined by sequencing the deposited plasmid in accordance with known methods

[0015] The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular plasmid can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.

[0016] Also provided in Table 1A is the name of the vector which contains the cDNA plasmid. Each vector is routinely used in the art. The following additional information is provided for convenience.

[0017] Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Phagemid pBS may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excised from the Zap Express vector. Both phagemids may be transformed into E. coli strain XL-1 Blue, also available from Stratagene.

[0018] Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, Inc., P. O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from Life Technologies. See, for instance, Gruber, C. E., et al., Focus 15:59 (1993). Vector lafmid BA (Bento Soares, Columbia University, New York, N.Y.) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).

[0019] The present invention also relates to the genes corresponding to SEQ ID NO: X, SEQ ID NO: Y, and/or a deposited cDNA (Clone ID). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include, but are not limited to, preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.

[0020] Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO: X and SEQ ID NO: Y using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.

[0021] The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO: X and/or a cDNA contained in ATCC Deposit No. Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO: Y, a polypeptide encoded by SEQ ID NO: X, and/or a polypeptide encoded by a cDNA contained in ATCC deposit No. Z. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO: Y, a polypeptide encoded by SEQ ID NO: X and/or a polypeptide encoded by the cDNA contained in ATCC Deposit No. Z, are also encompassed by the invention. The present invention further encompasses a polynucleotide comprising, or alternatively consisting of the complement of the nucleic acid sequence of SEQ ID NO: X, and/or the complement of the coding strand of the cDNA contained in ATCC Deposit No. Z.

[0022] Description of Table 1B

[0023] Table 1B summarizes some of the polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID), contig sequences (contig identifier (Contig ID:) and contig nucleotide sequence identifier (SEQ ID NO: X)) and further summarizes certain characteristics of these polynucleotides and the polypeptides encoded thereby. The first column provides the gene number in the application for each clone identifier. The second column provides a unique clone identifier, “Clone ID”, for a cDNA clone related to each contig sequence disclosed in Table 1A and/or 1B. The third column provides a unique contig identifier, “Contig ID:” for each of the contig sequences disclosed in Table 1B. The fourth column provides the sequence identifier, “SEQ ID NO: X”, for each of the contig sequences disclosed in Table 1A and/or 1B. The fifth column, “ORF (From-To)”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO: X that delineate the preferred open reading frame (ORF) that encodes the amino acid sequence shown in the sequence listing and referenced in Table 1B as SEQ ID NO: Y (column 6). Column 7 lists residues comprising predicted epitopes contained in the polypeptides encoded by each of the preferred ORFs (SEQ ID NO: Y). Identification of potential immunogenic regions was performed according to the method of Jameson and Wolf (CABIOS, 4; 181-186 (1988)); specifically, the Genetics Computer Group (GCG) implementation of this algorithm, embodied in the program PEPTIDESTRUCTURE (Wisconsin Package v10.0, Genetics Computer Group (GCG), Madison, Wis.). This method returns a measure of the probability that a given residue is found on the surface of the protein. Regions where the antigenic index score is greater than 0.9 over at least 6 amino acids are indicated in Table 1B as “Predicted Epitopes”. In particular embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more of the predicted epitopes described in Table 1B. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly. Column 8, “Tissue Distribution” shows the expression profile of tissue, cells, and/or cell line libraries which express the polynucleotides of the invention. The first number in column 8 (preceding the colon), represents the tissue/cell source identifier code corresponding to the key provided in Table 4. Expression of these polynucleotides was not observed in the other tissues and/or cell libraries tested. For those identifier codes in which the first two letters are not “AR”, the second number in column 8 (following the colon), represents the number of times a sequence corresponding to the reference polynucleotide sequence (e.g., SEQ ID NO: X) was identified in the tissue/cell source. Those tissue/cell source identifier codes in which the first two letters are “AR” designate information generated using DNA array technology. Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines. Probe synthesis was performed in the presence of ³³P dCTP, using oligo(dT) to prime reverse transcription. After hybridization, high stringency washing conditions were employed to remove non-specific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A local background signal subtraction was performed before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after “[array code]:” represents the mean of the duplicate values, following background subtraction and probe normalization. One of skill in the art could routinely use this information to identify normal and/or diseased tissue(s) which show a predominant expression pattern of the corresponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression. Column 9 provides the chromosomal location of polynucleotides corresponding to SEQ ID NO: X. Chromosomal location was determined by finding exact matches to EST and cDNA sequences contained in the NCBI (National Center for Biotechnology Information) UniGene database. Given a presumptive chromosomal location, disease locus association was determined by comparison with the Morbid Map, derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.) 2000. World Wide Web URL: http:/www.ncbi.nlm.nih.gov/omim/). If the putative chromosomal location of the Query overlaps with the chromosomal location of a Morbid Map entry, an OMIM identification number is disclosed in column 10 labeled “OMIM Disease Reference(s)”. A key to the OMIM reference identification numbers is provided in Table 5.

[0024] Description of Table 1C

[0025] Table 1C summarizes additional polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO: X)), and genomic sequences (SEQ ID NO: B). The first column provides a unique clone identifier, “Clone ID”, for a cDNA clone related to each contig sequence. The second column provides the sequence identifier, “SEQ ID NO: X”, for each contig sequence. The third column provides a unique contig identifier, “Contig ID:” for each contig sequence. The fourth column, provides a BAC identifier “BAC ID NO:A” for the BAC clone referenced in the corresponding row of the table. The fifth column provides the nucleotide sequence identifier, “SEQ ID NO: B” for a fragment of the BAC clone identified in column four of the corresponding row of the table. The sixth column, “Exon From-To”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO: B which delineate certain polynucleotides of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof).

[0026] Description of Table 1D

[0027] Table 1D: In preferred embodiments, the present invention encompasses a method of treating a disease or disorder listed in the “FEATURES OF PROTEIN” sections (below) and also as listed in the “Preferred Indications” column of Table 1D (below); comprising administering to a patient in which such treatment, prevention, or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) represented by Table 1A and Table 1D (in the same row as the disease or disorder to be treated is listed in the “Preferred Indications” column of Table 1D) in an amount effective to treat, prevent, or ameliorate the disease or disorder.

[0028] As indicated in Table 1D, the polynucleotides, polypeptides, agonists, or antagonists of the present invention (including antibodies) can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists thereof (including antibodies) could be used to treat the associated disease.

[0029] The present invention encompasses methods of preventing, treating, diagnosing, or ameliorating a disease or disorder. In preferred embodiments, the present invention encompasses a method of treating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient in which such treatment, prevention, or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to treat, prevent, diagnose, or ameliorate the disease or disorder. The first and second columns of Table 1D show the “Gene No.” and “Clone ID”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in preventing, treating, diagnosing, or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in Column 3 of Table 1D.

[0030] In another embodiment, the present invention also encompasses methods of preventing, treating, diagnosing, or ameliorating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in Column 3 of Table 1D.

[0031] The “Preferred Indication” column describes diseases, disorders, and/or conditions that may be treated, prevented, diagnosed, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).

[0032] The recitation of “Cancer” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., leukemias, cancers, and/or as described below under “Hyperproliferative Disorders”).

[0033] In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D may be used for example, to diagnose, treat, prevent, and/or ameliorate a neoplasm located in a tissue selected from the group consisting of: colon, abdomen, bone, breast, digestive system, liver, pancreas, prostate, peritoneum, lung, blood (e.g., leukemia), endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), uterus, eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.

[0034] In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a pre-neoplastic condition, selected from the group consisting of: hyperplasia (e.g., endometrial hyperplasia and/or as described in the section entitled “Hyperproliferative Disorders”), metaplasia (e.g., connective tissue metaplasia, atypical metaplasia, and/or as described in the section entitled “Hyperproliferative Disorders”), and/or dysplasia (e.g., cervical dysplasia, and bronchopulmonary dysplasia).

[0035] In another specific embodiment, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a benign dysproliferative disorder selected from the group consisting of: benign tumors, fibrocystic conditions, tissue hypertrophy, and/or as described in the section entitled “Hyperproliferative Disorders”.

[0036] The recitation of “Immune/Hematopoietic” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).

[0037] In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having the “Immune/Hematopoietic” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: anemia, pancytopenia, leukopenia, thrombocytopenia, leukemias, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, asthma, AIDS, autoimmnune disease, rheumatoid arthritis, granulomatous disease, immune deficiency, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, immune reactions to transplanted organs and tissues, systemic lupus erythematosis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and allergies.

[0038] The recitation of “Reproductive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the reproductive system (e.g., as described below under “Reproductive System Disorders”).

[0039] In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Reproductive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cryptorchism, prostatitis, inguinal hernia, varicocele, leydig cell tumors, verrucous carcinoma, prostatitis, malacoplakia, Peyronie's disease, penile carcinoma, squamous cell hyperplasia, dysmenorrhea, ovarian adenocarcinoma, Turner's syndrome, mucopurulent cervicitis, Sertoli-leydig tumors, ovarian cancer, uterine cancer, pelvic inflammatory disease, testicular cancer, prostate cancer, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, testicular atrophy, testicular feminization, anorchia, ectopic testis, epididymitis, orchitis, gonorrhea, syphilis, testicular torsion, vasitis nodosa, germ cell tumors, stromal tumors, dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding, cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, cervical neoplasms, pseudohermaphroditism, and premenstrual syndrome.

[0040] The recitation of “Musculoskeletal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the immune system (e.g., as described below under “Immune Activity”).

[0041] In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Musculoskeletal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bone cancers (e.g., osteochondromas, benign chondromas, chondroblastoma, chondromyxoid fibromas, osteoid osteomas, giant cell tumors, multiple myeloma, osteosarcomas), Paget's Disease, rheumatoid arthritis, systemic lupus erythematosus, osteomyelitis, Lyme Disease, gout, bursitis, tendonitis, osteoporosis, osteoarthritis, muscular dystrophy, mitochondrial myopathy, cachexia, and multiple sclerosis.

[0042] The recitation of “Cardiovascular” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”).

[0043] In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cardiovascular” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: myxomas, fibromas, rhabdomyomas, cardiovascular abnormalities (e.g., congenital heart defects, cerebral arteriovenous malformations, septal defects), heart disease (e.g., heart failure, congestive heart disease, arrhythmia, tachycardia, fibrillation, pericardial Disease, endocarditis), cardiac arrest, heart valve disease (e.g., stenosis, regurgitation, prolapse), vascular disease (e.g., hypertension, coronary artery disease, angina, aneurysm, arteriosclerosis, peripheral vascular disease), hyponatremia, hypernatremia, hypokalemia, and hyperkalemia.

[0044] The recitation of “Mixed Fetal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”).

[0045] In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Mixed Fetal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: spina bifida, hydranencephaly, neurofibromatosis, fetal alcohol syndrome, diabetes mellitus, PKU, Down's syndrome, Patau syndrome, Edwards syndrome, Turner syndrome, Apert syndrome, Carpenter syndrome, Conradi syndrome, Crouzon syndrome, cutis laxa, Cornelia de Lange syndrome, Ellis-van Creveld syndrome, Holt-Oram syndrome, Kartagener syndrome, Meckel-Gruber syndrome, Noonan syndrome, Pallister-Hall syndrome, Rubinstein-Taybi syndrome, Scimitar syndrome, Smith-Lemli-Opitz syndrome, thromocytopenia-absent radius (TAR) syndrome, Treacher Collins syndrome, Williams syndrome, Hirschsprung's disease, Meckel's diverticulum, polycystic kidney disease, Turner's syndrome, and gonadal dysgenesis, Klippel-Feil syndrome, Ostogenesis imperfecta, muscular dystrophy, Tay-Sachs disease, Wilm's tumor, neuroblastoma, and retinoblastoma.

[0046] The recitation of “Excretory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and renal disorders (e.g., as described below under “Renal Disorders”).

[0047] In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Excretory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bladder cancer, prostate cancer, benign prostatic hyperplasia, bladder disorders (e.g., urinary incontinence, urinary retention, urinary obstruction, urinary tract Infections, interstitial cystitis, prostatitis, neurogenic bladder, hematuria), renal disorders (e.g., hydronephrosis, proteinuria, renal failure, pyelonephritis, urolithiasis, reflux nephropathy, and unilateral obstructive uropathy).

[0048] The recitation of “Neural/Sensory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the nervous system (e.g., as described below under “Neural Activity and Neurological Diseases”).

[0049] In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Neural/Sensory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: brain cancer (e.g., brain stem glioma, brain tumors, central nervous system (Primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, and cerebral astrocytoma, neurodegenerative disorders (e.g., Alzheimer's Disease, Creutzfeldt-Jakob Disease, Parkinson's Disease, and Idiopathic Presenile Dementia), encephalomyelitis, cerebral malaria, meningitis, metabolic brain diseases (e.g., phenylketonuria and pyruvate carboxylase deficiency), cerebellar ataxia, ataxia telangiectasia, and AIDS Dementia Complex, schizophrenia, attention deficit disorder, hyperactive attention deficit disorder, autism, and obsessive compulsive disorders.

[0050] The recitation of “Respiratory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”).

[0051] In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Respiratory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of the respiratory system such as larynx cancer, pharynx cancer, trachea cancer, epiglottis cancer, lung cancer, squamous cell carcinomas, small cell (oat cell) carcinomas, large cell carcinomas, and adenocarcinomas. Allergic reactions, cystic fibrosis, sarcoidosis, histiocytosis X, infiltrative lung diseases (e.g., pulmonary fibrosis and lymphoid interstitial pneumonia), obstructive airway diseases (e.g., asthma, emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis and asbestosis), pneumonia, and pleurisy.

[0052] The recitation of “Endocrine” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”), renal disorders (e.g., as described below under “Renal Disorders”), and disorders of the endocrine system (e.g., as described below under “Endocrine Disorders”.

[0053] In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having an “Endocrine” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of endocrine tissues and organs (e.g., cancers of the hypothalamus, pituitary gland, thyroid gland, parathyroid glands, pancreas, adrenal glands, ovaries, and testes), diabetes (e.g., diabetes insipidus, type I and type II diabetes mellitus), obesity, disorders related to pituitary glands (e.g., hyperpituitarism, hypopituitarism, and pituitary dwarfism), hypothyroidism, hyperthyroidism, goiter, reproductive disorders (e.g. male and female infertility), disorders related to adrenal glands (e.g., Addison's Disease, corticosteroid deficiency, and Cushing's Syndrome), kidney cancer (e.g., hypernephroma, transitional cell cancer, and Wilm's tumor), diabetic nephropathy, interstitial nephritis, polycystic kidney disease, glomerulonephritis (e.g., IgM mesangial proliferative glomerulonephritis and glomerulonephritis caused by autoimmune disorders; such as Goodpasture's syndrome), and nephrocalcinosis.

[0054] The recitation of “Digestive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the gastrointestinal system (e.g., as described below under “Gastrointestinal Disorders”.

[0055] In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Digestive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: ulcerative colitis, appendicitis, Crohn's disease, hepatitis, hepatic encephalopathy, portal hypertension, cholelithiasis, cancer of the digestive system (e.g., biliary tract cancer, stomach cancer, colon cancer, gastric cancer, pancreatic cancer, cancer of the bile duct, tumors of the colon (e.g., polyps or cancers), and cirrhosis), pancreatitis, ulcerative disease, pyloric stenosis, gastroenteritis, gastritis, gastric atropy, benign tumors of the duodenum, distension, irritable bowel syndrome, malabsorption, congenital disorders of the small intestine, bacterial and parasitic infection, megacolon, Hirschsprung's disease, aganglionic megacolon, acquired megacolon, colitis, anorectal disorders (e.g., anal fistulas, hemorrhoids), congenital disorders of the liver (e.g., Wilson's disease, hemochromatosis, cystic fibrosis, biliary atresia, and alpha1-antitrypsin deficiency), portal hypertension, cholelithiasis, and jaundice.

[0056] The recitation of “Connective/Epithelial” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), and or to promote or inhibit regeneration (e.g., as described below under “Regeneration”), and wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”).

[0057] In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Connective/Epithelial” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: connective tissue metaplasia, mixed connective tissue disease, focal epithelial hyperplasia, epithelial metaplasia, mucoepithelial dysplasia, graft v. host disease, polymyositis, cystic hyperplasia, cerebral dysplasia, tissue hypertrophy, Alzheimer's disease, lymphoproliferative disorder, Waldenstron's macroglobulinemia, Crohn's disease, pernicious anemia, idiopathic Addison's disease, glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, cystic fibrosis, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, osteoporosis, osteocarthritis, periodontal disease, wound healing, relapsing polychondritis, vasculitis, polyarteritis nodosa, Wegener's granulomatosis, cellulitis, rheumatoid arthritis, psoriatic arthritis, discoid lupus erythematosus, systemic lupus erythematosus, scleroderma, CREST syndrome, Sjogren's syndrome, polymyositis, dermatomyositis, mixed connective tissue disease, relapsing polychondritis, vasculitis, Henoch-Schonlein syndrome, erythema nodosum, polyarteritis nodosa, temporal (giant cell) arteritis, Takayasu's arteritis, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, cellulitis, keloids, Ehler Danlos syndrome, Marfan syndrome, pseudoxantoma elasticum, osteogenese imperfecta, chondrodysplasias, epidermolysis bullosa, Alport syndrome, and cutis laxa.

[0058] Description of Table 1E

[0059] Table 1E provides information related to biological activities and preferred indications for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Table 1E also provides information related to assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column (“Gene No.”) provides the gene number in the application for each clone identifier. The second column (“Clone ID”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B, 1C, and 1D. The third column (“AA SEQ ID NO: Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A, 1B, and 2). The fourth column (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides). The fifth column (“Exemplary Activity Assay”) further describes the corresponding biological activity and provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The sixth column (“Preferred Indications”) describes particular embodiments of the invention and indications (e.g. pathologies, diseases, disorders, abnormalities, etc.) for which polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) may be used in detecting, diagnosing, preventing, and/or treating.

[0060] Table 1E describes the use of FMAT technology, inter alia, for testing or demonstrating various biological activities. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based system which provides a means to perform nonradioactive cell- and bead-based assays to detect activation of cell signal transduction pathways. This technology was designed specifically for ligand binding and immunological assays. Using this technology, fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using a data processing system. Unbound flurophore comprising the background signal is ignored, allowing for a wide variety of homogeneous assays. FMAT technology may be used for peptide ligand binding assays, immunofluorescence, apoptosis, cytotoxicity, and bead-based immunocapture assays. See, Miraglia S et. al., “Homogeneous cell and bead based assays for highthroughput screening using flourometric microvolume assay technology,” Journal of Biomolecular Screening; 4:193-204 (1999). In particular, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides (including polypeptide fragments and variants) to activate signal transduction pathways. For example, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides to upregulate production of immunomodulatory proteins (such as, for example, interleukins, GM-CSF, Rantes, and Tumor Necrosis factors, as well as other cellular regulators (e.g. insulin)).

[0061] Table 1E also describes the use of kinase assays for testing, demonstrating, or quantifying biological activity. In this regard, the phosphorylation and de-phosphorylation of specific amino acid residues (e.g. Tyrosine, Serine, Threonine) on cell-signal transduction proteins provides a fast, reversible means for activation and de-activation of cellular signal transduction pathways. Moreover, cell signal transduction via phosphorylation/de-phosphorylation is crucial to the regulation of a wide variety of cellular processes (e.g. proliferation, differentiation, migration, apoptosis, etc.). Accordingly, kinase assays provide a powerful tool useful for testing, confirming, and/or identifying polypeptides (including polypeptide fragments and variants) that mediate cell signal transduction events via protein phosphorylation. See e.g., Forrer, P., Tamaskovic R., and Jaussi, R. “Enzyme-Linked Immunosorbent Assay for Measurement of JNK, ERK, and p38 Kinase Activities” Biol. Chem. 379(8-9): 1101-1110 (1998).

[0062] Description of Table 2

[0063] Table 2 summarizes homology and features of some of the polypeptides of the invention. The first column provides a unique clone identifier, “Clone ID”, corresponding to a cDNA clone disclosed in Table 1A or 1B. The second column provides the unique contig identifier, “Contig ID:” corresponding to contigs in Table 1B and allowing for correlation with the information in Table 1B. The third column provides the sequence identifier, “SEQ ID NO: X”, for the contig polynucleotide sequence. The fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined. Comparisons were made between polypeptides encoded by the polynucleotides of the invention and either a non-redundant protein database (herein referred to as “NR”), or a database of protein families (herein referred to as “PFAM”) as further described below. The fifth column provides a description of the PFAM/NR hit having a significant match to a polypeptide of the invention. Column six provides the accession number of the PFAM/NR hit disclosed in the fifth column. Column seven, “Score/Percent Identity”, provides a quality score or the percent identity, of the hit disclosed in columns five and six. Columns 8 and 9, “NT From” and “NT To” respectively, delineate the polynucleotides in “SEQ ID NO: X” that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth and sixth columns. In specific embodiments polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence encoded by a polynucleotide in SEQ ID NO: X as delineated in columns 8 and 9, or fragments or variants thereof.

[0064] Description of Table 3

[0065] Table 3 provides polynucleotide sequences that may be disclaimed according to certain embodiments of the invention. The first column provides a unique clone identifier, “Clone ID”, for a cDNA clone related to contig sequences disclosed in Table 1B. The second column provides the sequence identifier, “SEQ ID NO: X”, for contig sequences disclosed in Table 1A and/or 1B. The third column provides the unique contig identifier, “Contig ID:”, for contigs disclosed in Table 1B. The fourth column provides a unique integer ‘a’ where ‘a’ is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO: X, and the fifth column provides a unique integer ‘b’ where ‘b’ is any integer between 15 and the final nucleotide of SEQ ID NO: X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: X, and where b is greater than or equal to a+14. For each of the polynucleotides shown as SEQ ID NO: X, the uniquely defined integers can be substituted into the general formula of a−b, and used to describe polynucleotides which may be preferably excluded from the invention. In certain embodiments, preferably excluded from the invention are at least one, two, three, four, five, ten, or more of the polynucleotide sequence(s) having the accession number(s) disclosed in the sixth column of this Table (including for example, published sequence in connection with a particular BAC clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequence(s) contained in the clones corresponding to at least one, two, three, four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC clone).

[0066] Description of Table 4

[0067] Table 4 provides a key to the tissue/cell source identifier code disclosed in Table 1B, column 8. Column 1 provides the tissue/cell source identifier code disclosed in Table 1B, Column 8. Columns 2-5 provide a description of the tissue or cell source. Codes corresponding to diseased tissues are indicated in column 6 with the word “disease”. The use of the word “disease” in column 6 is non-limiting. The tissue or cell source may be specific (e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a normal portion of a diseased organ). Furthermore, tissues and/or cells lacking the “disease” designation may still be derived from sources directly or indirectly involved in a disease state or disorder, and therefore may have a further utility in that disease state or disorder. In numerous cases where the tissue/cell source is a library, column 7 identifies the vector used to generate the library.

[0068] Description of Table 5

[0069] Table 5 provides a key to the OMIM reference identification numbers disclosed in Table 1B, column 10. OMIM reference identification numbers (Column 1) were derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine, (Bethesda, Md.) 2000. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/). Column 2 provides diseases associated with the cytologic band disclosed in Table 1B, column 9, as determined using the Morbid Map database.

[0070] Definitions

[0071] The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

[0072] In the present invention, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term “isolated” does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.

[0073] In the present invention, a “secreted” protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a “mature” protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.

[0074] As used herein, a “polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NO: X (as described in column 5 of Table 1A), or cDNA clone (as described in column 2 of Table 1A and contained within a pool of plasmids deposited with the ATCC in ATCC Deposit No: Z). For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5′ and 3′ untranslated sequences, the coding region, with or without a natural or artificial signal sequence, the protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a “polypeptide” refers to a molecule having an amino acid sequence encoded by a polynucleotide of the invention as broadly defined (obviously excluding poly-Phenylalanine or poly-Lysine peptide sequences which result from translation of a polyA tail of a sequence corresponding to a cDNA).

[0075] In the present invention, a representative plasmid containing the sequence of SEQ ID NO: X was deposited with the American Type Culture Collection (“ATCC”) and/or described in Table 1A. As shown in Table 1A, each cDNA is identified by a cDNA clone identifier and the ATCC Deposit Number (ATCC Deposit No: Z). Plasmids that were pooled and deposited as a single deposit have the same ATCC Deposit Number. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.

[0076] A “polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO: X, or the complement thereof (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments described herein) and/or sequences of the cDNA contained in the deposited clone (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments described herein). “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C. in a solution comprising 50% fornamide, 5× SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1× SSC at about 65 degree C.

[0077] Also included within “polynucleotides” of the present invention are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C. in a solution comprising 6× SSPE (20× SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA; followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5× SSC).

[0078] Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0079] Of course, a polynucleotide which hybridizes only to polyA+sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of “polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).

[0080] The polynucleotides of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.

[0081] In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

[0082] “SEQ ID NO: X” refers to a polynucleotide sequence described in column 5 of Table 1A, while “SEQ ID NO: Y” refers to a polypeptide sequence described in column 10 of Table 1A. SEQ ID NO: X is identified by an integer specified in column 6 of Table 1A. The polypeptide sequence SEQ ID NO: Y is a translated open reading frame (ORF) encoded by polynucleotide SEQ ID NO: X. The polynucleotide sequences are shown in the sequence listing immediately followed by all of the polypeptide sequences. Thus, a polypeptide sequence corresponding to polynucleotide sequence SEQ ID NO: 2 is the first polypeptide sequence shown in the sequence listing. The second polypeptide sequence corresponds to the polynucleotide sequence shown as SEQ ID NO: 3, and so on.

[0083] The polypeptides of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992)).

[0084] The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

[0085] The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.

[0086] The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the polypeptides of the present invention in methods which are well known in the art.

[0087] By a polypeptide demonstrating a “functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein of the invention. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide for binding) to an anti-polypeptide antibody], immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide.

[0088] “A polypeptide having functional activity” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular assay, such as, for example, a biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention).

[0089] The functional activity of the polypeptides, and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.

[0090] For example, in one embodiment where one is assaying for the ability to bind or compete with full-length polypeptide of the present invention for binding to an antibody to the full length polypeptide, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

[0091] In another embodiment, where a ligand is identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E., et al., Microbiol. Rev. 59:94-123 (1995). In another embodiment, physiological correlates polypeptide of the present invention binding to its substrates (signal transduction) can be assayed. In addition, assays described herein (see Examples) and otherwise known in the art may routinely be applied to measure the ability of polypeptides of the present invention and fragments, variants derivatives and analogs thereof to elicit polypeptide related biological activity (either in vitro or in vivo). Other methods will be known to the skilled artisan and are within the scope of the invention.

[0092] Polynucleotides and Polypeptides of the Invention

[0093] Features of Protein Encoded by Gene No: 1

[0094] The translation product of this gene shares sequence homology with several human sodium-dependent phosphate transporters (See e.g., Genebank Acc. Nos. gi|2062692 and gi|450532), and a rabbit renal cortical Na/Pa-i-cotransporter which is thought to be important in cellular metabolism and kidney function (See Genbank Accession No. gi|165690).

[0095] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence:

[0096] HELGGLLADFLLSRKILRLITIRKLFTAIGVLFPSVILVSLPWVRSSHSMTMTFL VLSSAISSFCESGALVNFLDIAPRYTGFLKGLLQVFAHIAGAISPTAAGFFISQD SEFGWRNVFLLSAAVNISGLVFYLIFGRAD VQDWAKEQTFTHL (SEQ ID NO: 123); HELGGLLADFLLSRKILRLITI (SEQ ID NO: 125);

[0097] RKLFTAIGVLFPSVILVSL PWVRS (SEQ ID NO: 126);

[0098] SHSMTMTFLVLSSAISSFCESGAL (SEQ ID NO: 127);

[0099] VNFLDIAPRYTGFLKGLLQVFAH (SEQ ID NO: 128;

[0100] IAGAISPTAAGFFISQDSEF GWRN (SEQ ID NO: 129);

[0101] VFLLSAAVNISGLVFYLIFGRADVQDWA KEQTFTHL (SEQ ID NO: 130);

[0102] LMKNPAAVGEMAPAMCAKTCNSPLRKPVYRGAISKKLTRA PDSQKLLMAEDSTKKVMVMLWLDLTQGRDTRITDGKRTPMAVKSFLMVMS LR IFLERRKSASRPPSSC (SEQ ID NO: 124);

[0103] LMKNPAAVGEMAPAMCAKTCNSPL RKPV (SEQ ID NO: 131);

[0104] YRGAISKKLTRAPDSQKLLMAED STKKVMVM (SEQ ID NO: 132); and/or

[0105] LWLDLTQGRDTRITDGKRTPMAVKSFLMVMSLRIFLERR KSASRPPSSC (SEQ ID NO: 133). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0106] This gene is expressed primarily in human adult small intestine.

[0107] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, renal and gastrointestinal disorders, or other disorders where ion homeostasis is aberrant. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the renal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., renal, neural, gastrointestinal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, bile, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0108] The tissue distribution in small intestine combined with the homology to a family of Sodium-dependent phosphate transporters suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study, diagnosis, treatment, and/or prevention of renal and gastointestinal disorders and/or diseases. The protein can also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions and as nutritional supplements. It may also have a very wide range of biological activities. Typical of these are cytokine, cell proliferation/differentiation modulating activity or induction of other cytokines;

[0109] immunostimulating/immunosuppressant activities (e.g., for treating human immunodeficiency virus infection, cancer, autoimmune diseases and allergy); regulation of hematopoiesis (e.g., for treating anaemia or as adjunct to chemotherapy); stimulation or growth of bone, cartilage, tendons, ligaments and/or nerves (e.g., for treating wounds, stimulation of follicle stimulating hormone (for control of fertility); chemotactic and chemokinetic activities (e.g., for treating infections, tumors); hemostatic or thrombolytic activity (e.g., for treating haemophilia, cardiac infarction etc.); anti-inflammatory activity (e.g., for treating septic shock, Crohn's disease); as antimicrobials; for treating psoriasis or other hyperproliferative diseases; for regulation of metabolism, and behavior. Also contemplated is the use of the corresponding nucleic acid in gene therapy procedures. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0110] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 11 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 666 of SEQ ID NO: 11, b is an integer of 15 to 680, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 11, and where b is greater than or equal to a+14.

[0111] Features of Protein Encoded by Gene No: 2

[0112] The translation product of this gene shares sequence homology with several members of a recently described subfamily of P-type ATPases, these ATPases are thought to play a general role in ATP-dependent aminophospholipid transport. Members of this subfamily include, the human FIC1 PROTEIN (see Genbank Accession No. gi|3628757) which is thought to play an essential role in enterohepatic circulation of bile acids, as it's deletion has been linked to Cholestasis, or impaired bile flow. Other members include, the putative E1-E2 ATPase from Mus musculus, which is thought to be important in phagocytosis, blood clotting, and cellular metabolism (See Genbank Accession No. gi|2895095 (AF011337)), and Bovine and Murine chromaffin granule ATPase II homologs (see Genbank Accession No. gi|4115341 and gi|1663648)

[0113] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: EYSTPDTVHLRKTILFSVKVPVLSEKMYCICPKSSVMFR ARHCSCESVSSSYNCMSWLMKYTWHALTISMEXYKEMGSKPAELYHVKNE LTAAVTGDKELPSDLGT (SEQ ID NO: 134); NQGSAEQQWAPLQAXKLERQ (SEQ ID NO: 135); YSSAGFDPISLYXSIEIVKACQVYFINQDMQLYDEETDS QLQCRALNITEDLGQIQYIFSDKTGTLTENKMVFRRCTVSGVEYSHDANEGLL RDAQWSTRLAGSISISFSGLLTGPCCFDSAPCLCLKF (SEQ ID NO: 137); YSSAGFDPISLYXSIEIVKAC QVYFI (SEQ ID NO: 138); NQDMQLYDEETDSQ LQCRALNITEDL (SEQ ID NO: 139); GQIQYIFSDKTGTLTENKMVFRRCTVSG (SEQ ID NO: 140); VEYSHD ANEGLLRDAQWSTRLAGSISIS (SEQ ID NO: 141); FSGLLTGPCCFDSAPCLCL KF (SEQ ID NO: 142); and/or IRHETLRNTDAXXG IVIYAGHETKALLNNSGPRY KRXSWRGR (SEQ ID NO: 136). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0114] The gene encoding the disclosed cDNA is believed to reside on chromosome 15. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 15.

[0115] This gene is expressed primarily in human prostate.

[0116] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, reproductive or cellular metabolism disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, reproductive, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, seminal fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell samples taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0117] The tissue distribution in prostate tissue combined with the homology to members of a conserved subfamily of P-type ATPases suggests that polynucleotides and polypeptides corresponding to this gene are useful for treatment and diagnosis of reproductive, metabolic, or haemopoietic disorders, such as congenital afflictions or proliferative conditions, including cancers. The expression in the prostate tissue may indicate the gene or its products can be used in disorders of the prostate, including inflammatory disorders, such as chronic prostatitis, granulomatous prostatitis and malacoplakia, prostatic hyperplasia and prostate neoplastic disorders, including adenocarcinoma, transitional cell carcinomas, ductal carcinomas, squamous cell carcinomas, or as hormones or factors with systemic or reproductive functions. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0118] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 12 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 727 of SEQ ID NO: 12, b is an integer of 15 to 741, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 12, and where b is greater than or equal to a+14.

[0119] Features of Protein Encoded by Gene No: 3

[0120] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: HELGPVCLHAIMLAELIFLFRSLHGILASAGTIGAVAAWL (SEQ ID NO: 143). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0121] This gene is expressed primarily in human testes.

[0122] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, reproductive disorders, particularly of the testes. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the male reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., reproductive, testes, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, seminal fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0123] The tissue distribution in testes suggests that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis, treatment, and/or prevention of various reproductive or endocrine disorders, such as male infertility and hormone imbalances. Similarly, the tissue distribution in testes indicates that the protein product of this clone is useful for the treatment and diagnosis of conditions concerning normal testicular function (e.g., endocrine function, sperm maturation), as well as cancer. Therefore, this gene product is useful in the treatment of male infertility and/or impotence. This gene product is also useful in assays designed to identify binding agents, as such agents (antagonists) are useful as male contraceptive agents.

[0124] The secreted protein may also be used as a contraceptive. Similarly, the protein is believed to be useful in the treatment and/or diagnosis of testicular cancer. The testes are also a site of active gene expression of transcripts that may be expressed, particularly at low levels, in other tissues of the body. Therefore, this gene product may be expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0125] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 13 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 605 of SEQ ID NO: 13, b is an integer of 15 to 619, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 13, and where b is greater than or equal to a+14.

[0126] Features of Protein Encoded by Gene No: 4

[0127] The translation product of this clone has been shown to have homology to the Y isoform of the conserved ubiquitous human TPR motif which is thought to be important in maintaining cellular metabolism in addition to a possible connection in increasing an individual's susceptibility to male infertility (See Genbank Accession No. gi|2580574 (AF000994)). In addition, the translation product of this clone shows homology to the murine male-specific histocompatibility antigen H-YDb (See Genbank Accession No. gnl|PID|e300472) from which is derived an epitope of the complex male-specific transplantation antigen H—Y.

[0128] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: DFGTXSDPKLFEMIKYCLLKILKQYQTLREALVAAGKEV IWHGRTNDEPAHYCSICEVEVFNLLFVTNESNTQKTYIVHCHDCARKTSKSLE NFVVLEQYKMEDLIQVYDQFTLASPWPPMDQSAFTSSLLRPIKALGSGRAEQ TSGDQLQKGATHSRASSLLRAAEMTRRPASREELPDPGLFCHSIKLLFVLL (SEQ ID NO: 144); DFGTXSDPKLFEMIKYCLLKILKQYQ (SEQ ID NO: 145); TLREALVAAGKEVIWHGRTNDEPAHYCS (SEQ ID NO: 146); ICEVEVFNLLF VTNESNTQKTYIVHC (SEQ ID NO: 147); HDCARKTSKSLENFVVLEQYKMED LIQVYD (SEQ ID NO: 148); QFTLASPWPPMDQSAFTSSLLRPIKALGSG (SEQ ID NO: 149); RAEQTSGDQLQKGATHSRASSLLRAAEMT (SEQ ID NO: 150); and/or RRPASREELPDPGLFCHSIKLLFVLL (SEQ ID NO: 151). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0129] This gene is expressed primarily in activated human neutrophils and synovium.

[0130] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune or hematopoieitic disorders, particularly inflammatory conditions, and/or autoimmune disorders, such as arthritis. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the inflammatory and immune systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, skeletal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0131] The tissue distribution in neutrophils and synovium, in addition to the homology of the translated product of this clone to a murine male-specific histocompatibility antigen, suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study, diagnosis, and treatment of inflammatory and immune disorders. Moreover, the protein may be useful for the detection and/or treatment of disorders and conditions afflicting the skeletal system, in particular osteoporosis, bone cancer, connective tissue disorders, (e.g., arthritis, trauma, tendonitis, chrondomalacia and inflammation). The protein is also useful in the diagnosis or treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis, dwarfism, spinal deformation, joint abnormalities, and chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0132] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 14 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 597 of SEQ ID NO: 14, b is an integer of 15 to 611, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 14, and where b is greater than or equal to a+14.

[0133] Features of Protein Encoded by Gene No: 5

[0134] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: LPGNFRPPRVILTFQWRFYLSFRKL (SEQ ID NO: 152); XIPPXXLPGNFRPPRVILTFQWRFYLSFRKL (SEQ ID NO: 154); and/or YLLLPCGLLSFWMCGALVVSPFVQNGQGQRLREARSLCLLKGTTWIFLMLSL PHFLVQELKFSNNFFSTVVIFSTSGFLQPTLIFLKLSWKSTHL (SEQ ID NO: 153). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0135] This gene is expressed primarily in anergic T-cells.

[0136] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune or hematopoietic disorders, particularly inflammatory or immunodeficiency conditions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid, and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0137] The tissue distribution in T-cells suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study and treatment of immune defects. More specifically, the gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses).

[0138] Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0139] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 15 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 571 of SEQ ID NO: 15, b is an integer of 15 to 585, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 15, and where b is greater than or equal to a+14.

[0140] Features of Protein Encoded by Gene No: 6

[0141] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: YMMVHCKYSVYNLLNKWIGFSIFPHWTWIDLEIGGLNL QVEIKGPNNCRVAGEG RYKCSKGGSR (SEQ ID NO: 155). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0142] This gene is expressed primarily in anergic T-cells.

[0143] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune and hemopoietic disorders, particularly inflammatory or immunodeficiency conditions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0144] The tissue distribution in T-cells suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study and treatment of inflammation, and other immune and hematopoietic disorders. Specifically, the gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0145] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 16 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1026 of SEQ ID NO: 16, b is an integer of 15 to 1040, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 16, and where b is greater than or equal to a+14.

[0146] Features of Protein Encoded by Gene No: 7

[0147] When tested against U937 Myeloid cell lines, supernatants removed from cells containing this gene activated the GAS assay. Thus, it is likely that this gene activates myeloid cells through the Jak-STAT signal transduction pathway. The gamma activating sequence (GAS) is a promoter element found upstream of many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.

[0148] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: MSAALWTYMRFLACLNHSSGSMYLSVNSTPVLLLLLV PNSARARAEFLQPGGXTSSRAAXXAVELQLLFPLXXG (SEQ ID NO: 156); FRQARNLMYVHNAADIHSSLPQHITVISPRELCHTFSLLKPATLDLLCSLSVGN LFRISERQCKH (SEQ ID NO: 157); and/or RVNVSSIMDIHEVPGLSKSQLWFN VPVCQLHTCVAVAARAEFGTSSCRIPAARGXH (SEQ ID NO: 158). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0149] This gene is expressed primarily in prostate cancer.

[0150] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, reproductive disorders, particular prostate cancer or infertility. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the prostate, especially of the reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., reproductive, prostate, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, seminal fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0151] The tissue distribution in prostate cancer, along with activation of the GAS assay when supernatants of cells containing this gene were tested against U937 Myeloid cell lines, suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of disorders of the reproductive system, particular proliferative conditions such as cancer. Similarly, the gene or its products can be used in the disorders of the prostate, including inflammatory disorders, such as chronic prostatitis, granulomatous prostatitis and malacoplakia, prostatic hyperplasia and prostate neoplastic disorders, including adenocarcinoma, transitional cell carcinomas, ductal carcinomas, squamous cell carcinomas, or as hormones or factors with systemic or reproductive functions. Protein, as well as, antibodies directed against the protein may show utility as a tissue-specific marker and/or immunotherapy target for the above listed tissues.

[0152] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 17 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 611 of SEQ ID NO: 17, b is an integer of 15 to 625, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 17, and where b is greater than or equal to a+14.

[0153] Features of Protein Encoded by Gene No: 8

[0154] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: IRHEGNSCTNKTAHAVLTASYTECSC (SEQ ID NO: 159). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0155] This gene is expressed primarily in prostate.

[0156] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, disorders of the reproductive system, particularly cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., reproductive, prostate, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, seminal fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0157] The tissue distribution in prostate suggests that polynucleotides and polypeptides corresponding to this gene are useful for treatment and diagnosis of disorders of the reproductive system, such as proliferative conditions, including, but not limited to, prostate cancer. Similarly, the gene or its products can be used in the treatment of disorders of the prostate, including inflammatory disorders, such as chronic prostatitis, granulomatous prostatitis and malacoplakia, prostatic hyperplasia and prostate neoplastic disorders, including adenocarcinoma, transitional cell carcinomas, ductal carcinomas, squamous cell carcinomas, or as hormones or factors with systemic or reproductive functions. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0158] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 18 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 805 of SEQ ID NO: 18, b is an integer of 15 to 819, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 18, and where b is greater than or equal to a+14.

[0159] Features of Protein Encoded by Gene No: 9

[0160] This gene is expressed primarily in anergic T-cells.

[0161] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune and blood disorders, particularly inflammatory or immunodeficiency conditions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoeitic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0162] The tissue distribution in T-cells suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study and treatment of immune and inflammatory disorders. Moreover, the gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses).

[0163] Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0164] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 19 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 768 of SEQ ID NO: 19, b is an integer of 15 to 782, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 19, and where b is greater than or equal to a+14.

[0165] Features of Protein Encoded by Gene No: 10

[0166] This gene is expressed primarily in anergic T-cells.

[0167] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune or hematopoietic disorders, particularly inflammatory conditions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0168] The tissue distribution in T-cells suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study and treatment of immune and blood disorders. Moreover, this gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity, immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and tissues.

[0169] In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0170] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 20 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 641 of SEQ ID NO: 20, b is an integer of 15 to 655, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 20, and where b is greater than or equal to a+14.

[0171] Features of Protein Encoded by Gene No: 11

[0172] When tested against K562 cell lines, supernatants removed from cells containing this gene activated the ISRE (interferon-sensitive responsive element) pathway. Thus, it is likely that this gene activates leukemia cells, and to a lesser extent, in immune and hematopoietic cells and tissues, through the Jak-STAT signal transduction pathway. ISRE is a promoter element found upstream in many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells.

[0173] This gene is expressed primarily in smooth muscle.

[0174] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, vascular disorders, particularly microvascular disease, atherosclerosis, aneurysm, and stroke. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the vascular and smooth muscle tissues, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g, vascular tissue, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0175] The tissue distribution in smooth muscle, combined with the detected ISRE biological activity, suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study, diagnosis, and treatment of vascular disorders. The protein product of this gene is useful for the diagnosis, treatment, and/or prevention of conditions and pathologies of the cardiovascular system, such as heart disease, restenosis, atherosclerosis, stoke, angina, thrombosis, and wound healing. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0176] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 21 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 784 of SEQ ID NO: 21, b is an integer of 15 to 798, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 21, and where b is greater than or equal to a+14.

[0177] Features of Protein Encoded by Gene No: 12

[0178] The translation product of this gene shares sequence homology with the human IL-8 receptor (PF4AR), which is thought to be important in inflammatory disorders.

[0179] This gene is expressed primarily in synovial sarcoma.

[0180] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, inflammatory disorders such as rheumatoid arthritis. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, skeletal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0181] The tissue distribution in synovial sarcoma, combined with the homology to an IL-8 receptor (PF4AR), suggests that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis, treatment, and/or prevention of inflammatory disorders such as rheumatoid arthritis. Similarly, this gene product may also be useful for the detection and treatment of osteoporosis, bone cancer, as well as, disorders afflicting connective tissues (e.g., arthritis, trauma, tendonitis, chrondomalacia and inflammation), such as in the diagnosis or treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid).

[0182] The secreted protein can also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, and as nutritional supplements. It may also have a very wide range of biological activities. Typical of these are cytokine, cell proliferation/differentiation modulating activity or induction of other cytokines; immunostimulating/immunosuppressant activities (e.g. for treating human immunodeficiency virus infection, cancer, autoimmune diseases and allergy); regulation of hematopoiesis (e.g. for treating anemia or as adjunct to chemotherapy); stimulation or growth of bone, cartilage, tendons, ligaments and/or nerves (e.g. for treating wounds, stimulation of follicle stimulating hormone (for control of fertility); chemotactic and chemokinetic activities (e.g. for treating infections, tumors); hemostatic or thrombolytic activity (e.g. for treating hemophilia, cardiac infarction etc.); anti-inflammatory activity (e.g. for treating septic shock, Crohn's disease); as antimicrobials; for treating psoriasis or other hyperproliferative diseases; for regulation of metabolism, and behavior. Also contemplated is the use of the corresponding nucleic acid in gene therapy procedures. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0183] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 22 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 632 of SEQ ID NO: 22, b is an integer of 15 to 646, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 22, and where b is greater than or equal to a+14.

[0184] Features of Protein Encoded by Gene No: 13

[0185] When tested against PC12 cell lines, supernatants removed from cells containing this gene activated the EGR1 (early growth response gene 1) promoter element. Thus, it is likely that this gene activates sensory neuron cells, and to a lesser extent, in neural cells and tissues, through the EGR1 signal transduction pathway. EGR1 is a separate signal transduction pathway from Jak-STAT. Genes containing the EGR1 promoter element are induced in various tissues and cell types upon activation, leading the cells to undergo differentiation and proliferation.

[0186] This gene is expressed primarily in human testes.

[0187] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, reproductive disorders, particularly afflictions of the testes, such as male infertility and testicular cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., reproductive, testes, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, seminal fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0188] The tissue distribution in testes combined with the detected EGR1 biological activity suggests that polynucleotides and polypeptides corresponding to this gene are useful for various reproductive disorders such as male infertility or associated endocrine disorders. Similarly, the protein product of this clone is useful for the treatment and diagnosis of conditions concerning proper testicular function (e.g., endocrine function, sperm maturation), as well as cancer. Therefore, this gene product is useful in the treatment of male infertility and/or impotence. This gene product is also useful in assays designed to identify binding agents, as such agents (antagonists) are useful as male contraceptive agents. Similarly, the protein is believed to be useful in the treatment and/or diagnosis of testicular cancer. The testes are also a site of active gene expression of transcripts that may be expressed, particularly at low levels, in other tissues of the body. Therefore, this gene product may be expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0189] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 23 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 738 of SEQ ID NO: 23, b is an integer of 15 to 752, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 23, and where b is greater than or equal to a+14.

[0190] Features of Protein Encoded by Gene No: 14

[0191] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence:

[0192] YKVVLVWREDQSSHKIHLSQTLIQNKALTLFNSMKAERGEEAXGKNVSS (SEQ ID NO: 160); YKVVLVWREDQSSHKIHLSQTLIQ (SEQ ID NO: 162); NKALTLFNSMKAERGEEAXGKNVSS (SEQ ID NO: 163); DGELSKCCMCSD YTIDCYFPISLPLLGRPYYLRHNIEIRPYINHTMMASKGSSKRMGCTSFTLTQKLE IIILSEKGMWKAEIGQKLGXLHHS (SEQ ID NO: 161); DGELSKCCMCSDYTIDC YFPISLPLLGRPYY (SEQ ID NO: 164); LRHNIEIRPYINHTMASKGSSKRMGCT SFTLT (SEQ ID NO: 165); and/or QKLEIIILSEKGMWKAEIGQKLGXLHHS (SEQ ID NO: 166). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0193] This gene is expressed primarily in fetal bone and testical tumors.

[0194] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, developmental, skeletal, or reproductive disorders, particularly, proliferative conditions such as testicular cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the male reproductive systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., reproductive, skeletal, testes, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, seminal fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0195] The tissue distribution in testes suggests that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis, treatment, and/or prevention of testicular cancer. In addition, expression of this gene product in the testis may implicate this gene product in normal testicular function. In addition, this gene product may be useful in the treatment of male infertility, and/or could be used as a male contraceptive.

[0196] Moreover, this gene product may be useful in the detection and treatment of disorders and conditions afflicting the skeletal system, in particular osteoporosis, bone cancer, as well as, disorders afflicting connective tissues (e.g., arthritis, trauma, tendonitis, chrondomalacia and inflammation), such as in the diagnosis or treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias (ie. spondyloepiphysial dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphysial chondrodysplasia type Schmid).

[0197] Expression within fetal tissue and other cellular sources marked by proliferating cells suggests that this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of cancer and other proliferative disorders. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Thus, this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0198] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 24 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 801 of SEQ ID NO: 24, b is an integer of 15 to 815, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 24, and where b is greater than or equal to a+14.

[0199] Features of Protein Encoded by Gene No: 15

[0200] This gene is expressed primarily in stomach.

[0201] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, gastrointestinal and digestive disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the gastrointestinal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., gastrointestinal, stomach, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, bile, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0202] The tissue distribution in stomach tissue suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study and treatment of gastrointestinal, digestive, and general metabolic disorders. For example, the protein product of this clone may be useful for the diagnosis, prevention, and/or treatment of various metabolic disorders such as Tay-Sach's disease, phenylkenonuria, galactosemia, porphyrias, and Hurler's syndrome. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0203] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 25 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 864 of SEQ ID NO: 25, b is an integer of 15 to 878, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 25, and where b is greater than or equal to a+14.

[0204] Features of Protein Encoded by Gene No: 16

[0205] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: MLCINVQTHVYECA (SEQ ID NOP: 167). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0206] This gene is expressed primarily in anergic T-cells and CD34 depleted cord blood.

[0207] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, hemopoietic, immune, or reproductive conditions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., hemopoietic, immune, or reproductive, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0208] The tissue distribution in T-cells suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study and treatment of immune, inflammatory and other hematopoietic disorders. In addition, polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of hematopoetic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages.

[0209] The uses include bone marrow cell ex vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia. The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0210] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 26 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 836 of SEQ ID NO: 26, b is an integer of 15 to 850, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 26, and where b is greater than or equal to a+14.

[0211] Features of Protein Encoded by Gene No: 17

[0212] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: LCCPGWSAVVRSWLTATLASWVQAILMDSASQVAGIT SVHHQAQLSFVFLVEMGLCHVGQAGLKLLASSDLPASASQSAGITGMSHHS WPERTSFIFKI (SEQ ID NO: 168); LCCPGWSAVVRSWLTATLASWVQAILMD SASQ (SEQ ID NO: 169); VAGITSVHHQAQLSFVFLVEMGLCHVGQAGLKLLA (SEQ ID NO: 170); and/or SSDLPASASQSAGITGMSHHSWPERTSFIFKI (SEQ ID NO: 171). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0213] This gene is expressed primarily in human adult small intestine.

[0214] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, gastrointestinal disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the gastrointestinal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., gastrointestinal, metabolic, and cancerous and wounded tissues) or bodily fluids (e.g., bile, lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0215] The tissue distribution in small intestine suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study, diagnosis, and treatment of gastrointestinal disorders, and/or treatment of various metabolic disorders such as Tay-Sachs disease, phenylkenonuria, galactosemia, porphyrias, and Hurler's syndrome. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0216] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 27 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 774 of SEQ ID NO: 27, b is an integer of 15 to 788, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 27, and where b is greater than or equal to a+14.

[0217] Features of Protein Encoded by Gene No: 18

[0218] The translation product of this gene was shown to have homology to the F33H2.2 protein from Caenorhabditis elegans (See Genbank Accession No. gnl|PID|e1297838). Considering the homology to a C. elegans protein, an important and vital function may be attributed to this clone based upon its conservation.

[0219] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: FGRGNTILFLRHNKDLVAQTAQPDQPNYGFPLDLLRCES LLGLDPATCSRVLNKNYTLLVSMAPLTNEIRPVSSCTPQHIGPAIPEVSSVWFK LYIYHVTGQGPPSLLLSKGTRLRKLPDIFQSYDRLXITSWGHDPGVVPTSNVL TMLNDALTHSAVLIQGHGLHGIGETVHVPFPFDETELQGEFTRVNMGVHKAL QILRNRVXLQHLCGYVTMLNASSQLADRKLSDASDERGEPDLASGSDVNGST ESFEMVIEEATIDSATKQTSGATTEADWVP LV (SEQ ID NO: 172); FGRGNTIL FLRHNKDLVAQTAQPDQPNYGFPLDLLRCESLLGLDPATCSRVLNKNYTLLV SMAPLTNEIRPVSSCTPQHIGPAIPEVSSVWFKLYIYHVTGQGPPSLLLSKGTR LRKLPDIFQSYDRLXITSWGHDPGVVPTSNVLTMLNDALTHSAVLIQGHGLH GIGETVHVP (SEQ ID NO: 173); LRHNKDLVAQTAQPDQPNYGF(SEQ ID NO: 174); FPLDLLRCESLLGLDPATCSR (SEQ ID NO: 175); RVLNKNYTLL VSMAPLTNEIR (SEQ ID NO: 176); R PVSSCTPQHIGPAIPEVSS (SEQ ID NO: 177); SVWFKLYIYHVTGQGPPSLLL (SEQ ID NO: 178); LSKGTRLRKLPDIF QSYDRLX (SEQ ID NO: 179); XITSWGHDPGVV PTSNVLTM (SEQ ID NO: 180); MLNDALTHSAVLIQGHGLHGI(SEQ ID NO: 181); FPFDETELQGEFTRV NMGVHKALQILRNRVXLQHLCGYVTMLNASSQLADRKLSDASDERGEPDLA SGSDVNGSTESFEMVIEEATIDSATKQTSGATTEADWVP LV (SEQ ID NO: 182); GEFTRVNMGVHKALQILRNRV (SEQ ID NO: 183); VXLQHLCGYVTML NASSQLA (SEQ ID NO: 184); ADRKLSDASDERGEPDLASGS (SEQ ID NO: 185); LRKLHSQTNPI (SEQ ID NO: 187); and/or SDVNGSTESFEMVIEEATIDS (SEQ ID NO: 186). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0220] This gene is expressed primarily in healing groin wound, and to a lesser extent, in synovium.

[0221] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, developmental, wound healing, and synovial disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the synovium and epithelium, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., developmental, skeletal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0222] The tissue distribution in wounded tissue suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study, diagnosis, and treatment of disorders involving the synovium and epithelium. Specifically, polynucleotides and polypeptides corresponding to this gene are useful for the treatment, diagnosis, and/or prevention of various skin disorders including congenital disorders (i.e. nevi, moles, freckles, Mongolian spots, hemangiomas, port-wine syndrome), integumentary tumors (i.e. keratoses, Bowen's disease, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, Paget's disease, mycosis fungoides, and Kaposi's sarcoma), injuries and inflammation of the skin (i.e. wounds, rashes, prickly heat disorder, psoriasis, dermatitis), atherosclerosis, uticaria, eczema, photosensitivity, autoimmune disorders (i.e. lupus erythematosus, vitiligo, dermatomyositis, morphea, scleroderma, pemphigoid, and pemphigus), keloids, striae, erythema, petechiae, purpura, and xanthelasma. In addition, such disorders may predispose an individual viral and bacterial infections of the skin (i.e. cold sores, warts, chickenpox, molluscum contagiosum, herpes zoster, boils, cellulitis, erysipelas, impetigo, tinea, althletes foot, and ringworm).

[0223] Moreover, the protein product of this clone may also be useful for the treatment or diagnosis of various connective tissue disorders such as arthritis, trauma, tendonitis, chrondomalacia and inflammation, autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). The protein is useful in the detection, treatment, and/or prevention of skeletal diseases and disorders, which include, but are not limited to osteoporosis, bone cancer, etc. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0224] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 28 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 824 of SEQ ID NO: 28, b is an integer of 15 to 838, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 28, and where b is greater than or equal to a+14.

[0225] Features of Protein Encoded by Gene No: 19

[0226] This gene is expressed primarily in ovarian cancer.

[0227] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, disorders of the reproductive system, particularly proliferative disorders of the ovary, such as cancer and cysts. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., reproductive, ovarian tissue, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0228] The tissue distribution in ovarian tissue suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of disorders of the reproductive system and cancers. Moreover, the expression within cellular sources marked by proliferating cells (i.e., ovarian cancer tissues) suggests this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, cancer, and other proliferative conditions. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation.

[0229] Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and would be useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0230] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 29 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 741 of SEQ ID NO: 29, b is an integer of 15 to 755, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 29, and where b is greater than or equal to a+14.

[0231] Features of Protein Encoded by Gene No: 20

[0232] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: RNFYLYFLPYCVVCVC (SEQ ID NO: 188). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0233] This gene is expressed primarily in prostate cancer.

[0234] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, reproductive disorders, particularly proliferative conditions of the prostate, such as cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the reproductive disorders and cancer, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., reproductive, prostate, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, seminal fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0235] The tissue distribution in prostate suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of disorders of the reproductive organs and cancers, such as male infertility. Protein may also be useful as a contraceptive. Moreover, the expression within cellular sources marked by proliferating cells (i.e., prostate cancer) suggests this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, cancer, and other proliferative conditions. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation.

[0236] Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and would be useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. The protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0237] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 30 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 799 of SEQ ID NO: 30, b is an integer of 15 to 813, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 30, and where b is greater than or equal to a+14.

[0238] Features of Protein Encoded by Gene No: 21

[0239] This gene is expressed primarily in B-cells and rhabdomyosarcoma.

[0240] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune, hematopoietic, or muscular disorders, particularly proliferative conditions such as cancer or fibroids. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the haemopoietic and muscular system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, muscular, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0241] The tissue distribution in immune and muscle tissues or cells suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of a variety of disorders, such as for the detection, and/or prevention of muscular dystrophy, cardiomyopathy, fibroids, myomas, and rhabdomyosarcomas. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and tissues.

[0242] Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0243] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 31 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 499 of SEQ ID NO: 31, b is an integer of 15 to 513, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 3 1, and where b is greater than or equal to a+14.

[0244] Features of Protein Encoded by Gene No: 22

[0245] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: GTRSINLLFFRCILEGGKSVEEQLCNSYKFS (SEQ ID NO: 189). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0246] This gene is expressed primarily in anergic T-cells.

[0247] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune and blood conditions, particularly inflammatory or immunodeficiency disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0248] The tissue distribution in T-cells suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study and treatment of inflammation and immune disorders. More specifically, the gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0249] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 32 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 562 of SEQ ID NO: 32, b is an integer of 15 to 576, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 32, and where b is greater than or equal to a+14.

[0250] Features of Protein Encoded by Gene No: 23

[0251] The translation product of this gene was shown to have homology to the gi|2501808 brain digoxin carrier protein of Rattus norvegicus which is thought to serve the role as a sodium-independent organic anion transporter. This gene may also play a role in hormone transport. When tested against U937 cell lines, supernatants removed from cells containing this gene activated the GAS (gamma activating sequence) promoter element. Thus, it is likely that this gene activates promyelocytic cells, and to a lesser extent in other immune or hematopoietic cells and tissues, through the Jak-STAT signal transduction pathway. GAS is a promoter element found upstream of many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.

[0252] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: LTVPRRCPAATETNVDGQKVYRDCSCIPQNLSSGFGHA TAGXMHFNLSEKAPPSGFHIRCEFSLHSXSSIPALTATLRCVRDPQRSFALGIQ WIVVRILGGIPGPIAFGWVIDKACLLWQXQCGQXGSCLVYQXRP (SEQ ID NO: 190); VSLCHAGALQPRRR (SEQ ID NO: 191); ATETNVDGQKVYRDCSC IPQN (SEQ ID NO: 192); NLSSGFGHATAGXMHFNLSEK (SEQ ID NO: 193); KAPPSGFHIRCEFSLHSXSSI (SEQ ID NO: 194); IPALTATLRCVRDPQRSFAL (SEQ ID NO: 195); and/or LGIQWIVVRILGGIPGPIAFG (SEQ ID NO: 196). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0253] This gene is expressed primarily in retina.

[0254] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, visual disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the eye, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., opthalmic tissue, retinal tissue, cancerous and wounded tissues) or bodily fluids (e.g., lymph, vitreous humor, aqueous humor, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0255] The tissue distribution in retinal tissue combined with the detected GAS biological activity suggests that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis, treatment, and/or prevention of visual disorders of the eye. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0256] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 33 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1005 of SEQ ID NO: 33, b is an integer of 15 to 1019, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 33, and where b is greater than or equal to a+14.

[0257] Features of Protein Encoded by Gene No: 24

[0258] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: GTAHLPTLHWKPLLS (SEQ ID NO: 197). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0259] This gene is expressed primarily in synovial fluid of a patient with chronic synovitis.

[0260] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune or skeletal disorders, particularly inflammatory disorders such as arthritis. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, skeletal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0261] The tissue distribution in synovial fluid suggests that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis, treatment, and/or prevention of inflammatory disorders such as arthritis. Moreover, the expression of this gene product in synovium would suggest a role in the detection and treatment of disorders and conditions afflicting the skeletal system, in particular osteoporosis, bone cancer, connective tissue disorders (e.g., trauma, tendonitis, chrondomalacia and inflammation). The protein is also useful in the diagnosis or treatment of various autoimmune disorders (i.e., rheumatoid arthritis, lupus, scleroderma, and dermatomyositis), dwarfism, spinal deformation, joint abnormalities, and chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0262] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 34 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 419 of SEQ ID NO: 34, b is an integer of 15 to 433, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 34, and where b is greater than or equal to a+14.

[0263] Features of Protein Encoded by Gene No: 25

[0264] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: LVMQCLGQVLSPLRTSVCLPIERGRWPGMVPHTTSAL GG (SEQ ID NO: 198); EPACLSH (SEQ ID NO: 200); and/or QNTIHSLLPQGRM TKSLVLEEQKRKAGRSEMKLELLMRVSLWYSGQALVLLGLITNLSCSVLGKS FHLSGPLSVSL (SEQ ID NO: 199). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0265] This gene is expressed primarily in human synovium.

[0266] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune and skeletal diseases and/or disorders, particularly inflammatory conditions, such as arthritis. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and inflammatory systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., skeletal, synovium, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0267] The tissue distribution in synovium suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study, diagnosis, and treatment of immune and inflammatory disorders. In addition, the gene product may play a role in the detection and treatment of disorders and conditions affecting the skeletal system, in particular osteoporosis, bone cancer, connective tissue disorders (e.g., arthritis, trauma, tendonitis, chrondomalacia and inflammation). The protein is also useful in the diagnosis or treatment of autoimmune disorders which include rheumatoid arthritis, lupus, scleroderma, dermatomyositis, dwarfism, spinal deformation, joint abnormalities, and chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0268] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 35 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 628 of SEQ ID NO: 35, b is an integer of 15 to 642, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 35, and where b is greater than or equal to a+14.

[0269] Features of Protein Encoded by Gene No: 26

[0270] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: RDFGCEPSPGTDTGSLSFLV (SEQ ID NO: 201). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0271] This gene is expressed primarily in synovial fluid of a patient with chronic synovitis.

[0272] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune or skeletal disorders, particularly inflammatory disorders such as arthritis. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, skeletal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0273] The tissue distribution in synovium suggests that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis, treatment, and/or prevention of inflammatory disorders such as arthritis. In addition, the gene product may play a role in the detection and treatment of disorders and conditions afflicting the skeletal system, in particular osteoporosis, bone cancer, as well as, disorders of the connective tissues (e.g., arthritis, trauma, tendonitis, chrondomalacia and inflammation). Moreover, the protein is also useful in the diagnosis or treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, dermatomyositis, dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0274] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 36 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 653 of SEQ ID NO: 36, b is an integer of 15 to 667, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 36, and where b is greater than or equal to a+14.

[0275] Features of Protein Encoded by Gene No: 27

[0276] When tested against K562 cell lines, supernatants removed from cells containing this gene activated the ISRE (interferon-sensitive responsive element) promoter element. Thus, it is likely that this gene activates leukemia cells through the Jak-STAT signal transduction pathway. ISRE is a promoter element found upstream in many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells.

[0277] This gene is expressed primarily in anergic T-cells.

[0278] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune and blood disorders, particularly inflammatory or immunodeficiency disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0279] The tissue distribution in T-cells, combined with the detected ISRE biological activity suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study and treatment of immune and blood diseases. More specifically, the gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0280] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 37 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 640 of SEQ ID NO: 37, b is an integer of 15 to 654, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 37, and where b is greater than or equal to a+14.

[0281] Features of Protein Encoded by Gene No: 28

[0282] When tested against K562 cell lines, supernatants removed from cells containing this gene activated the ISRE (interferon-sensitive responsive element) promoter element. Thus, it is likely that this gene activates leukemia cells, and to a lesser extent, in immune and hematopoeitic cells and tissues, through the Jak-STAT signal transduction pathway. ISRE is a promoter element found upstream in many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells.

[0283] This gene is expressed primarily in human fibrosarcoma.

[0284] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, muscle disorders, particularly proliferative conditions such as cancer or fibroids. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the metabolic system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., muscle, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0285] The tissue distribution in fibrosarcoma, combined with the detected ISRE biological activity, suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study, diagnosis, and/or treatment of various cancers. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0286] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 38 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 717 of SEQ ID NO: 38, b is an integer of 15 to 731, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 38, and where b is greater than or equal to a+14.

[0287] Features of Protein Encoded by Gene No: 29

[0288] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: SVILLCPFF (SEQ ID NO: 202). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0289] This gene is expressed primarily in salivary gland.

[0290] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, epithelial, immune, and digestive disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the epithelial, digestive, and immune systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., epithelial, immune, digestive tissues, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0291] The tissue distribution in salivary tissue suggests that polynucleotides and polypeptides corresponding to this gene are useful for the study, diagnosis, and treatment of various disorders of the immune, digestive and epithelial systems. The protein is useful in modulating the immune response to aberrant polypeptides, as may be present in the cells and tissues of proliferative organs. The protein may also be useful in enhancing or inhibiting the bodies antibiological and microbial defenses (i.e., by enhancing the secretion of an antibiological agent in saliva, such as nitric oxide, for example). Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0292] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 39 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 364 of SEQ ID NO: 39, b is an integer of 15 to 378, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 39, and where b is greater than or equal to a+14.

[0293] Features of Protein Encoded by Gene No: 30

[0294] This gene is expressed primarily in spongy brain tissue obtained from Alzheimer's patients.

[0295] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, Alzheimer's disease; neurodegenerative disorders including, but not restricted to Alzheimer's, such as schizophrenia, ALS, etc. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the brain and nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0296] The tissue distribution suggests that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and/or treatment of neurodegenerative disorders, particularly Alzheimer's disease. Specific expression of this gene product in the brain tissue of Alzheimer's patients suggests that it may play a deleterious role in the progression of the disease. Alternately, it may represent an attempted response by the body to combat the progression of Alzheimer's. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0297] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 40 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 628 of SEQ ID NO: 40, b is an integer of 15 to 642, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 40, and where b is greater than or equal to a+14.

[0298] Features of Protein Encoded by Gene No: 31

[0299] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: ARDRTHCLL (SEQ ID NO: 203). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0300] This gene is expressed primarily in epididymus, and to a lesser extent, in colon tissue.

[0301] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, infertility; sperm developmental/survival disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types or cell types (e.g., reproductive, cancerous and wounded tissues) or bodily fluids (e.g., lymph, seminal fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0302] The tissue distribution in epididymus suggests that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and/or treatment of male infertility. Specific expression of this gene product within the epididymus suggests that it plays key roles in the development and/or survival of sperm. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0303] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 41 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 428 of SEQ ID NO: 41, b is an integer of 15 to 442, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 41, and where b is greater than or equal to a+14.

[0304] Features of Protein Encoded by Gene No: 32

[0305] This gene is expressed primarily in T-cells, osteoblasts, pancreas, colon and to a lesser extent, in healing groin wound library and many other tissues.

[0306] FIGS. 1A-C show the nucleotide (SEQ ID NO: 42) and deduced amino acid sequence (SEQ ID NO: 98) corresponding to this gene.

[0307]FIG. 2 shows an analysis of the amino acid sequence (SEQ ID NO: 98).

[0308] The data presented in FIG. 2 are also represented in tabular form in Table 6.

[0309] Also preferred are polypeptides comprising, or alternatively consisting of, the mature form of the polypeptide having the amino acid sequence of SEQ ID NO: 98, the mature form of the polypeptide encoded by the cDNA contained in ATCC Deposit No. 209277, and/or the mature polypeptide which is predicted to consist of residues 19-300 of the foregoing sequence (SEQ ID NO: 98), and biologically active fragments of these polypeptides (e.g., fragments that induce T-cell proliferation). Polynucleotides encoding these polypeptides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by nucleic acids which hybridize, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof are encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the inventions.

[0310] The present invention is further directed to fragments of the polynucleotide sequences described herein. By a fragment of, for example, the polynucleotide sequence of a deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 42, is intended polynucleotide fragments at least about 15 nt, and more preferably at least about 20 nt, at least about 25 nt, still more preferably at least about 30 nt, at least about 35 nt, and even more preferably, at least about 40 nt in length, at least about 45 nt in length, at least about 50 nt in length, at least about 60 nt in length, at least about 70 nt in length, at least about 80 nt in length, at least about 90 nt in length, at least about 100 nt in length, at least about 125 nt in length, at least about 150 nt in length, at least about 175 nt in length, which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 200-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of a deposited cDNA or as shown in SEQ ID NO: 42. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of a deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 42. In this context “about” includes the particularly recited size, an sizes larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150 from about 1151 to about 1200, from about 1201 to about 1250, from about 1251 to about 1300, from about 1301 to about 1350, from about 1351 to about 1400, from about 1401 to about 1450, and from about 1451 to about 1500, from about 1501 to about 1550, from about 1551 to about 1600 , from about 1 601 to about 1650 , from about 1651 to about 1734 of SEQ ID NO: 42, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attributes of the corresponding protein (e.g., the ability to induce T-cell proliferation).

[0311] Preferred polypeptide fragments of the invention comprise, or alternatively consist of, the secreted protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. Particularly, N-terminal deletions of the polypeptide can be described by the general formula m-300 where m is an integer from 2 to 294, where m corresponds to the position of the amino acid residue identified in SEQ ID NO: 98. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: K-2 to Q-300; F-3 to Q-300; L-4 to Q-300; L-5 to Q-300; D-6 to Q-300; I-7 to Q-300; L-8 to Q-300; L-9 to Q-300; L-10 to Q-300; L-11 to Q-300; P-12 to Q-300 ; L-13 to Q-300; L-14 to Q-300; I-15 to Q-300; V-16 to Q-300; C-17 to Q-300; S-18 to Q-300; E-20 to Q-300; S-21 to Q-300; F-22 to Q-300; V-23 to Q-300; K-24 to Q-300; L-25 to Q-300; F-26 to Q-300; I-27 to Q-300; P-28 to Q-300; K-29 to Q-300; R-30 to Q-300; R-31 to Q-300; K-32 to Q-300; S-33 to Q-300; V-34 to Q-300; T-35 to Q-300; G-36 to Q-300; E-37 to Q-300; I-38 to Q-300; V-39 to Q-300; L-40 to Q-300; I-41 to Q-300; T-42 to Q-300; G-43 to Q-300; A-44 to Q-300; G-45 to Q-300; H-46 to Q-300; G-47 to Q-300; I-48 to Q-300; G-49 to Q-300; R-50 to Q-300; L-51 to Q-300; T-52 to Q-300; A-53 to Q-300; Y-54 to Q-300; E-55 to Q-300; F-56 to Q-300; A-57 to Q-300; K-58 to Q-300; L-59 to Q-300; K-60 to Q-300; S-61 to Q-300; K-62 to Q-300; L-63 to Q-300; V-64 to Q-300; L-65 to Q-300; W-66 to Q-300; D-67 to Q-300; I-68 to Q-300; N-69 to Q-300; K-70 to Q-300; H-71 to Q-300; G-72 to Q-300; L-73 to Q-300; E-74 to Q-300; E-75 to Q-300; T-76 to Q-300; A-77 to Q-300; A-78 to Q-300; K-79 to Q-300; C-80 to Q-300; K-81 to Q-300; G-82 to Q-300; L-83 to Q-300; G-84 to Q-300; A-85 to Q-300; K-86 to Q-300; V-87 to Q-300; H-88 to Q-300; T-89 to Q-300; F-90 to Q-300; V-91 to Q-300; V-92 to Q-300; D-93 to Q-300; C-94 to Q-300; S-95 to Q-300; N-96 to Q-300; R-97 to Q-300; E-98 to Q-300; D-99 to Q-300; I-100 to Q-300; Y-101 to Q-300; S-102 to Q-300; S-103 to Q-300; A-104 to Q-300; K-105 to Q-300; K-106 to Q-300; V-107 to Q-300; K-108 to Q-300; A-109 to Q-300; E-110 to Q-300; I-111 to Q-300; G-112 to Q-300; D-113 to Q-300; V-114 to Q-300; S-115 to Q-300; I-116 to Q-300; L-117 to Q-300; V-118 to Q-300; N-119 to Q-300; N-120 to Q-300; A-121 to Q-300; G-122 to Q-300; V-123 to Q-300; V-124 to Q-300; Y-125 to Q-300; T-126 to Q-300; S-127 to Q-300; D-128 to Q-300; L-129 to Q-300; F-130 to Q-300; A-131 to Q-300; T-132 to Q-300; Q-133 to Q-300; D-134 to Q-300; P-135 to Q-300; Q-136 to Q-300; I-137 to Q-300; E-138 to Q-300; K-139 to Q-300; T-140 to Q-300; F-141 to Q-300; E-142 to Q-300; V-143 to Q-300; N-144 to Q-300; V-145 to Q-300; L-146 to Q-300; A-147 to Q-300; H-148 to Q-300; F-149 to Q-300; W-150 to Q-300; T-151 to Q-300; T-152 to Q-300; K-153 to Q-300; A-154 to Q-300; F-155 to Q-300; L-156 to Q-300; P-157 to Q-300; A-158 to Q-300; M-159 to Q-300; T-160 to Q-300; K-161 to Q-300; N-162 to Q-300; N-163 to Q-300; H-164 to Q-300; G-165 to Q-300; H-166 to Q-300; I-167 to Q-300; V-168 to Q-300; T-169 to Q-300; V-170 to Q-300; A-171 to Q-300; S-172 to Q-300; A-173 to Q-300; A-174 to Q-300; G-175 to Q-300; H-176 to Q-300; V-177 to Q-300; S-178 to Q-300; V-179 to Q-300; P-180 to Q-300; F-181 to Q-300; L-182 to Q-300; L-183 to Q-300; A-184 to Q-300; Y-185 to Q-300; C-186 to Q-300; S-187 to Q-300; S-188 to Q-300; K-189 to Q-300; F-190 to Q-300; A-191 to Q-300; A-192 to Q-300; V-193 to Q-300; G-194 to Q-300; F-195 to Q-300; H-196 to Q-300; K-197 to Q-300; T-198 to Q-300; L-199 to Q-300; T-200 to Q-300; D-201 to Q-300; E-202 to Q-300; L-203 to Q-300; A-204 to Q-300; A-205 to Q-300; L-206 to Q-300; Q-207 to Q-300; I-208 to Q-300; T-209 to Q-300; G-210 to Q-300; V-211 to Q-300; K-212 to Q-300; T-213 to Q-300; T-214 to Q-300; C-215 to Q-300; L-216 to Q-300; C-217 to Q-300; P-218 to Q-300; N-219 to Q-300; F-220 to Q-300; V-221 to Q-300; N-222 to Q-300; T-223 to Q-300; G-224 to Q-300; F-225 to Q-300; I-226 to Q-300; K-227 to Q-300; N-228 to Q-300; P-229 to Q-300; S-230 to Q-300; T-231 to Q-300; S-232 to Q-300; L-233 to Q-300; G-234 to Q-300; P-235 to Q-300; T-236 to Q-300; L-237 to Q-300; E-238 to Q-300; P-239 to Q-300; E-240 to Q-300; E-241 to Q-300; V-242 to Q-300; V-243 to Q-300; N-244 to Q-300; R-245 to Q-300; L-246 to Q-300; M-247 to Q-300; H-248 to Q-300; G-249 to Q-300; I-250 to Q-300; L-251 to Q-300; T-252 to Q-300; E-253 to Q-300; Q-254 to Q-300; K-255 to Q-300; M-256 to Q-300; I-257 to Q-300; F-258 to Q-300; I-259 to Q-300; P-260 to Q-300; S-261 to Q-300; S-262 to Q-300; I-263 to Q-300; A-264 to Q-300; F-265 to Q-300; L-266 to Q-300; T-267 to Q-300; T-268 to Q-300; L-269 to Q-300; E-270 to Q-300; R-271 to Q-300; I-272 to Q-300; L-273 to Q-300; P-274 to Q-300; E-275 to Q-300; R-276 to Q-300; F-277 to Q-300; L-278 to Q-300; A-279 to Q-300; V-280 to Q-300; L-281 to Q-300; K-282 to Q-300; R-283 to Q-300; K-284 to Q-300; I-285 to Q-300; S-286 to Q-300; V-287 to Q-300; K-288 to Q-300; F-289 to Q-300; D-290 to Q-300; A-291 to Q-300; V-292 to Q-300; I-293 to Q-300; G-294 to Q-300; and Y-295 to Q-300 ; of SEQ ID NO: 98. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by nucleic acids which hybridize, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof are encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the inventions. Antibodies that bind polypeptides of the invention are also encompassed by the invention. In preferred embodiments, the invention is directed to antibodies that bind and inhibit activity (e.g., T-cell proliferation).

[0312] Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification, of loss of one or more biological functions of the protein (e.g., ability to stimulate T-cell proliferation), other functional activities (e.g., biological activities, ability to multimerize, ability to bind ligand, ability to generate antibodies, ability to bind antibodies) may still be retained. For example the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a polypeptide with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.

[0313] Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in FIGS. 1A-C (SEQ ID NO: 98), as described by the general formula 1−n, where n is an integer from 6 to 299, where n corresponds to the position of the amino acid residue identified in SEQ ID NO: 98. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: M-1 to A-299; M-1 to K-298; M-1 to M-297; M-1 to K-296; M-1 to Y-295; M-1 to G-294; M-1 to I-293; M-1 to V-292; M-1 to A-291; M-1 to D-290; M-1 to F-289; M-1 to K-288; M-1 to V-287; M-1 to S-286; M-1 to 1-285; M-1 to K-284; M-1 to R-283; M-1 to K-282; M-1 to L-281; M-1 to V-280; M-1 to A-279; M-1 to L-278; M-1 to F-277; M-1 to R-276; M-1 to E-275; M-1 to P-274; M-1 to L-273; M-1 to I-272; M-1 to R-271; M-1 to E-270; M-1 to L-269; M-1 to T-268; M-1 to T-267; M-1 to L-266; M-1 to F-265; M-1 to A-264; M-1 to I-263; M-1 to S-262; M-1 to S-261; M-1 to P-260; M-1 to 1-259; M-1 to F-258; M-1 to I-257; M-1 to M-256; M-1 to K-255; M-1 Q-254; M-1 to E-253; M-1 to T-252; M-1 to L-251; M-1 to I-250; M-1 to G-249; M-1 H-248; M-1 to M-247; M-1 to L-246; M-1 to R-245; M-1 to N-244; M-1 to V-243; M-1 to V-242; M-1 to E-241; M-1 to E-240; M-1 to P-239; M-1 to E-238; M-1 to L237; M-1 to T-236; M-1 to P-235; M-1 to G-234; M-1 to L-233; M-1 to S-232; M-1 to T-231; M-1 to S-230; M-1 to P-229; M-1 to N-228; M-1 to K-227; M-1 to I-226; M-1 to F-225; M-1 to G-224; M-1 to T-223; M-1 to N-222; M-1 to V-221; M-1 to F-220; M-1 to N-219; M-1 to P-218; M-1 to C-217; M-1 to L-216; M-1 to C-215; M-1 to T-214; M-1 to T-213; M-1 to K-212; M-1 to V-211; M-1 to G-210; M-1 to T-209; M-1 to 1-208; M-1 to Q-207; M-1 to L-206; M-1 to A-205; M-1 to A-204; M-1 to L-203; M-1 to E-202; M-1 to D-201; M-1 to T-200; M-1 to L-199; M-1 to T-198; M-1 to K-197; M-1 to H-196; M-1 to F-195; M-1 to G-194; M-1 to V-193; M-1 to A-192; M-1 to A-191; M-1 to F-190; M-1 to K-189; M-1 to S-188; M-1 to S-187; M-1 to C-186; M-1 to Y-185; M-1 to A-184; M-1 to L-183; M-1 to L-182; M-1 to F-181; M-1 to P-180; M-1 to V-179; M-1 to S-178; M-1 to V-177; M-1 to H-176; M-1 to G-175; M-1 to A-174; M-1 to A-173; M-1 to S-172; M-1 to A-171; M-1 to V-170; M-1 to T-169; M-1 to V-168; M-1 to 1-167; M-1 to H-166; M-1 to G-165; M-1 to H-164; M-1 to N-163; M-1 to N-162; M-1 to K-161; M-1 to T-160; M-1 to M-159; M-1 to A-158; M-1 to P-157; M-1 to L-156; M-1 to F-155; M-1 to A-154; M-1 to K-153; M-1 to T-152; M-1 to T-151; M-1 to W-150; M-1 to F-149; M-1 to H-148; M-1 to A-147; M-1 to L-146; M-1 to V-145; M-1 to N-144; M-1 to V-143; M-I to E-142; M-1 to F-141; M-1 to T-140; M-1 to K-139; M-1 to E-138; M-1 to 1-137; M-1 to Q-136; M-1 to P-135; M-1 to D-134; M-1 to Q-133; M-1 to T-132; M-1 to A-131; M-1 to F-130; M-1 to L-129; M-1 to D-128; M-1 to S-127; M-1 to T-126; M-1 to Y-125; M-1 to V-124; M-1 to V-123; M-1 to G-122; M-1 to A-121; M-1 to N-120; M-1 to N-119; M-1 to V-118; M-1 to L-117; M-1 to I-116; M-1 to S-115; M-1 to V-114; M-1 to D-113; M-1 to G-112; M-1 to I-111; M-1 to E-110; M-1 to A-109; M-1 to K-108; M-1 to V-107; M-1 to K-106; M-1 to K-105; M-1 to A-104; M-1 to S-103; M-1 to S-102; M-1 to Y-101; M-1 to I-100; M-1 to D-99; M-1 to E-98; M-1 to R-97; M-1 to N-96; M-1 to S-95; M-1 to C-94; M-1 to D-93; M-1 to V-92; M-1 to V-91; M-1 to F-90; M-1 to T-89; M-1 to H-88; M-1 to V-87; M-1 to K-86; M-1 to A-85; M-1 to G-84; M-1 to L-83; M-1 to G-82; M-1 to K-81; M-1 to C-80; M-1 to K-79; M-1 to A-78; M-1 to A-77; M-1 to T-76; M-1 to E-75; M-1 to E-74; M-1 to L-73; M-1 to G-72; M-1 to H-71; M-1 to K-70; M-1 to N-69; M-1 to 1-68; M-1 to D-67; M-1 to W-66; M-1 to L-65; M-1 to V-64; M-1 to L-63; M-1 to K-62; M-1 to S-61; M-1 to K-60; M-1 to L-59; M-1 to K-58; M-1 to A-57; M-1 to F-56; M-1 to E-55; M-1 to Y-54; M-1 to A-53; M-1 to T-52; M-1 to L-51; M-1 to R-50; M-1 to G-49; M-1 to 1-48; M-1 to G-47; M-1 to H-46; M-1 to G-45; M-1 to A-44; M-1 to G-43; M-1 to T-42; M-1 to 1-41; M-1 to L-40; M-1 to V-39; M-1 to I-38; M-1 to E-37; M-1 to G-36; M-1 to T-35; M-1 to V-34; M-1 to S-33; M-1 to K-32; M-1 to R-31; M-1 to R-30; M-1 to K-29; M-1 to P-28; M-1 to I-27; M-1 to F-26; M-1 to L-25; M-1 to K-24; M-1 to V-23; M-1 to F-22; M-1 to S-21; M-1 to E-20; M-1 to L-19; M-1 to S-18; M-1 to C-17; M-1 to V-16; M-1 to I-15; M-1 to L-14; M-1 to L-13; M-1 to P-12; M-1 to L-11; M-1 to L-10; M-1 to L-9; M-1 to L-8; M-1 to I-7; and M-1 to D-6 of SEQ ID NO: 98. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by nucleic acids which hybridize, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof are encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the inventions. Antibodies that bind polypeptides of the invention are also encompassed by the invention. In preferred embodiments, the invention is directed to antibodies that bind and inhibit activity (e.g., T-cell proliferation).

[0314] In addition, any of the above listed N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides comprising, or alternatively consisting of, one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m−n of SEQ ID NO: 98, where n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0315] The present invention is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein as m−n. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N- and C-terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0316] Also included are polynucleotide sequences encoding a polypeptide consisting of a portion of the complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 209277, where this portion excludes any integer of amino acid residues from 1 to about 294 amino acids from the amino terminus of the complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 209277, or any integer of amino acid residues from 6 to about 300 amino acids from the carboxy terminus, or any combination of the above amino terminal and carboxy terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209277. Polypeptides encoded by these polynucleotides also are encompassed by the invention.

[0317] Additional preferred polypeptide fragments of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: M-1 to 1-15; K-2 to V-16; F-3 to C-17; L-4 to S-18; L-5 to L-19; D-6 to E-20; I-7 to S-21; L-8 to F-22; L-9 to V-23; L-10 to K-24; L-11 to L-25; P-12 to F-26; L-13 to I-27; L-14 to P-28; I-15 to K-29; V-16 to R-30; C-17 to R-31; S-18 to K-32; L-19 to S-33; E-20 to V-34; S-21 to T-35; F-22 to G-36; V-23 to E-37; K-24 to I-38; L-25 to V-39; F-26 to L-40; I-27 to I-41; P-28 to T-42; K-29 to G-43; R-30 to A-44; R-31 to G-45; K-32 to H-46; S-33 to G-47; V-34 to I-48; T-35 to G-49; G-36 to R-50; E-37 to L-51; I-38 to T-52; V-39 to A-53; L-40 to Y-54; I-41 to E-55; T-42 to F-56; G-43 to A-57; A-44 to K-58; G-45 to L-59; H-46 to K-60; G-47 to S-61; I-48 to K-62; G-49 to L-63; R-50 to V-64; L-51 to L-65; T-52 to W-66; A-53 to D-67; Y-54 to I-68; E-55 to N-69; F-56 to K-70; A-57 to H-71; K-58 to G-72; L-59 to L-73; K-60 to E-74; S-61 to E-75; K-62 to T-76; L-63 to A-77; V-64 to A-78; L-65 to K-79; W-66 to C-80; D-67 to K-81; I-68 to G-82; N-69 to L-83; K-70 to G-84; H-71 to A-85; G-72 to K-86; L-73 to V-87; E-74 to H-88; E-75 to T-89; T-76 to F-90; A-77 to V-91; A-78 to V-92; K-79 to D-93; C-80 to C-94; K-81 to S-95; G-82 to N-96; L-83 to R-97; G-84 to E-98; A-85 to D-99; K-86 to I-100; V-87 to Y-101; H-88 to S-102; T-89 to S-103; F-90 to A-104; V-91 to K-105; V-92 to K-106; D-93 to V-107; C-94 to K-108; S-95 to A-109; N-96 to E-110; R-97 to I-111; E-98 to G-112; D-99 to D-113; I-100 to V-1 14; Y-101 to S-115; S-102 to I-116; S-103 to L-117; A-104 to V-1 18; K-105 to N-119; K-106 to N-120; V-107 to A-121; K-108 to G-122; A-109 to V-123; E-110 to V-124; I-111 to Y-125; G-112 to T-126; D-113 to S-127; V-114 to D-128; S-115 to L-129; 1-116 to F-130; L-117 to A-131; V-118 to T-132; N-119 to Q-133; N-120 to D-134; A-121 to P-135; G-122 to Q-136; V-123 to I-137; V-124 to E-138; Y-125 to K-139; T-126 to T-140; S-127 to F-141; D-128 to E-142; L-129 to V-143; F-130 to N-144; A-131 to V-145; T-132 to L-146; Q-133 to A-147; D-134 to H-148; P-135 to F-149; Q-136 to W-150; I-137 to T-151; E-138 to T-152; K-139 to K-153; T-140 to A-154; F-141 to F-155; E-142 to L-156; V-143 to P-157; N-144 to A-158; V-145 to M-159; L-146 to T-160; A-147 to K-161; H-148 to N-162; F-149 to N-163; W-150 to H-164; T-151 to G-165; T-152 to H-166; K-153 to I-167; A-154 to V-168; F-155 to T-169; L-156 to V-170; P-157 to A-171; A-158 to S-172; M-159 to A-173; T-160 to A-174; K-161 to G-175; N-162 to H-176; N-163 to V-177; H-164 to S-178; G-165 to V-179; H-166 to P-180; I-167 to F-181; V-168 to L-182; T-169 to L-183; V-170 to A-184; A-171 to Y-185; S-172 to C-186; A-173 to S-187; A-174 to S-188; G-175 to K-189; H-176 to F-190; V-177 to A-191; S-178 to A-192; V-179 to V-193; P-180 to G-194; F-181 to F-195; L-182 to H-196; L-183 to K-197; A-184 to T-198; Y-185 to L-199; C-186 to T-200; S-187 to D-201; S-188 to E-202; K-189 to L-203; F-190 to A-204; A-191 to A-205; A-192 to L-206; V-193 to Q-207; G-194 to I-208; F-195 to T-209; H-196 to G-210; K-197 to V-211; T-198 to K-212; L-199 to T-213; T-200 to T-214; D-201 to C-215; E-202 to L-216; L-203 to C-217; A-204 to P-218; A-205 to N-219; L-206 to F-220; Q-207 to V-221; I-208 to N-222; T-209 to T-223; G-210 to G-224; V-211 to F-225; K-212 to I-226; T-213 to K-227; T-214 to N-228; C-215 to P-229; L-216 to S-230; C-217 to T-231; P-218 to S-232; N-219 to L-233; F-220 to G-234; V-221 to P-235; N-222 to T-236; T-223 to L-237; G-224 to E-238; F-225 to P-239; I-226 to E-240; K-227 to E-241; N-228 to V-242; P-229 to V-243; S-230 to N-244; T-231 to R-245; S-232 to L-246; L-233 to M-247; G-234 to H-248; P-235 to G-249; T-236 to I-250; L-237 to L-251; E-238 to T-252; P-239 to E-253; E-240 to Q-254; E-241 to K-255; V-242 to M-256; V-243 to I-257; N-244 to F-258; R-245 to I-259; L-246 to P-260; M-247 to S-261; H-248 to S-262; G-249 to I-263; I-250 to A-264; L-251 to F-265; T-252 to L-266; E-253 to T-267; Q-254 to T-268; K-255 to L-269; M-256 to E-270; I-257 to R-271; F-258 to I-272; 1-259 to L-273; P-260 to P-274; S-261 to E-275; S-262 to R-276; I-263 to F-277; A-264 to L-278; F-265 to A-279; L-266 to V-280; T-267 to L-281; T-268 to K-282; L-269 to R-283; E-270 to K-284; R-271 to I-285; I-272 to S-286; L-273 to V-287; P-274 to K-288; E-275 to F-289; R-276 to D-290; F-277 to A-291; L-278 to V-292; A-279 to I-293; V-280 to G-294; L-281 to Y-295; K-282 to K-296; R-283 to M-297; K-284 to K-298; I-285 to A-299; and S-286 to Q-300 of SEQ ID NO: 98. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by nucleic acids which hybridize, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof are encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the inventions. Antibodies that bind polypeptides of the invention are also encompassed by the invention. In preferred embodiments, the invention is directed to antibodies that bind and inhibit activity (e.g., T-cell proliferation).

[0318] Preferred polypeptides of the present invention comprise, or alternatively consist of, one, two, three, or all four of the immunogenic epitopes shown in SEQ ID NO: 98 as residues: Lys-29 to Val-34, Cys-94 to Asp-99, Ser-102 to Val-107, and Gln-133 to Lys-139. Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by nucleic acids which hybridize, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof are encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the inventions.

[0319] In additional nonexclusive embodiments, polypeptides of the invention, comprise or alternatively consist of, the following amino acid sequence: THQTLAATKG (SEQ ID NO: 204). Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind these polypeptides.

[0320]FIG. 2 shows an analysis of the amino acid sequence (SEQ ID NO: 98). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithyms. In the “Antigenic Index or Jameson-Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides.

[0321] The data presented in FIG. 2 are also represented in tabular form in Table 6. The columns are labeled with the headings “Res”, “Position”, and Roman Numerals I-XIV. The column headings refer to the following features of the amino acid sequence presented in FIG. 2, and Table 6: “Res”: amino acid residue of SEQ ID NO: 98 and FIGS. 1A-C; “Position”: position of the corresponding residue within SEQ ID NO: 98 and FIGS. 1A-C; I: Alpha, Regions—Garnier-Robson; II: Alpha, Regions—Chou-Fasman; III: Beta, Regions—Garnier-Robson; IV: Beta, Regions—Chou-Fasman; V: Turn, Regions—Garnier-Robson; VI: Turn, Regions—Chou-Fasman; VII: Coil, Regions—Garnier-Robson; VIII: Hydrophilicity Plot—Kyte-Doolittle; IX: Hydrophobicity Plot—Hopp-Woods; X: Alpha, Amphipathic Regions—Eisenberg; XI: Beta, Amphipathic Regions—Eisenberg; XII: Flexible Regions—Karplus-Schulz; XIII: Antigenic Index—Jameson-Wolf; and XIV: Surface Probability Plot—Emini.

[0322] Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consisting of, one or more of the following regions: alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attributes of the protein set forth in FIG. 2 and/or Table 6, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table 6 can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

[0323] Certain preferred regions in these regards are set out in FIG. 2, but may, as shown in Table 6, be represented or identified by using tabular representations of the data presented in FIG. 2. The DNA*STAR computer algorithm used to generate FIG. 2 (set on the original default parameters) was used to present the data in FIG. 2 in a tabular format (See Table 6). The tabular format of the data in FIG. 2 is used to easily determine specific boundaries of a preferred region.

[0324] The gene encoding the disclosed cDNA is thought to reside on chromosome 4. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 4.

[0325] As described herein or otherwise known in the art, the polynucleotides of the invention have uses that include, but are not limited to, serving as probes or primers in chromosome identification, chromosome mapping, and linkage analysis.

[0326] Additionally, polypeptides of the invention have been found induce T-cell proliferation. Representative examples of assays used to demonstrate T-cell proliferative activity are described in Example 33 herein.

[0327] Accordingly, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in the diagnosis, detection and/or treatment of diseases and/or immune disorders associated with aberrant T-cell proliferation and/or activation. In preferred embodiments, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in the diagnosis, detection and/or treatment of diseases and/or disorders relating to the immune system in general, and T-cell proliferation and/or activation specifically (e.g., cytokine production, inflammation, T cell proliferation and T cell proliferative disorders, and/or as described under “Immune Activity”, “Hyperproliferative Disorders” and “Diseases at the Cellular Level” below). Thus, polynucleotides, translation products and antibodies of the invention are useful in the diagnosis, detection and/or treatment of diseases and/or disorders associated with activities that include, but are not limited to, T cell activation, cytokine production, T cell proliferation, T cell proliferative disorders, T cell related immunodeficiencies (e.g., automimmune disorders), inflammation, graft rejection and immune system disorders.

[0328] Polynucleotides and polypeptides of the invention are also useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immunological and wound healing diseases and/or disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types or cell types (e.g., immune, metabolic, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0329] The tissue distribution in immune cells (e.g., T-cells) and the fact that polypeptides of this invention have been found induce T-cell proliferation indicates that polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention would be useful for the diagnosis, treatment, and/or prevention of a variety of immune system disorders. In particular, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing diseases, disorders, and/or conditions of the immune system, by, for example, activating or inhibiting the proliferation, activation, differentiation, or mobilization (chemotaxis) of immune cells. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing diseases, disorders, and/or conditions of hematopoietic cells. For example, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells. Examples of immunologic deficiency syndromes include, but are not limited to: blood protein diseases, disorders, and/or conditions (e.g., agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.

[0330] The expression in T-cells and the fact that polypeptides of this invention have been found to induce T-cell proliferation indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests a usefulness for treatment of cancer (e.g. by boosting immune responses). Expression in cells of lymphoid origin, indicates the natural gene product would be involved in immune functions. Therefore it would also be useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.

[0331] The tissue distribution in T-cells suggests that polynucleotides and polypeptides corresponding to this gene are useful for modulating the immune response during wound repair. The translation product of this gene may function as a stimulator for the growth of bone, cartilage, tendons, ligaments and/or nerves, which would be useful for the treatment of wounds. Moreover, the protein may be useful in detection, treating, and/or preventing metabolic conditions, particularly conditions related to aberrant fatty-acid metabolism. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues.

[0332] In specific non-exclusive embodiments, inflammatory conditions may also be treated, diagnosed, and/or prevented with polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention. Such inflammatory conditions include, but are not limited to, for example, respiratory disorders (such as, e.g., asthma and allergy); gastrointestinal disorders (such as, e.g., inflammatory bowel disease); cancers (such as, e.g., gastric, ovarian, lung, bladder, liver, and breast); CNS disorders (such as, e.g., multiple sclerosis, blood-brain barrier permeability, ischemic brain injury and/or stroke, traumatic brain injury, neurodegenerative disorders (such as, e.g., Parkinson's disease and Alzheimer's disease), AIDS-related dementia, and prion disease); cardiovascular disorders (such as, e.g., atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary bypass complications); as well as many additional diseases, conditions, and disorders that are characterized by inflammation (such as, e.g., chronic hepatitis (B and C), rheumatoid arthritis, gout, trauma, septic shock, pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusion injury, Grave's disease, systemic lupus erythematosis, diabetes mellitus (i.e., type 1 diabetes), and allogenic transplant rejection).

[0333] More specifically, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to treat, diagnose, and/or prevent transplantation rejections, graft-versus-host disease, autoimmune and inflammatory diseases (e.g., immune complex-induced vasculitis, glomerulonephritis, hemolytic anemia, myasthenia gravis, type II collagen-induced arthritis, experimental allergic and hyperacute xenograft rejection, rheumatoid arthritis, and systemic lupus erythematosus (SLE). Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. Polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.

[0334] Similarly, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may also be used to modulate and/or diagnose inflammation. For example, since polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists of the invention may inhibit the activation, proliferation and/or differentiation of cells involved in an inflammatory response, these molecules can be used to treat, diagnose, or prognose, inflammatory conditions, both chronic and acute conditions, including, but not limited to, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, and resulting from over production of cytokines (e.g., TNF or IL-1).

[0335] Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0336] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 42 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1720 of SEQ ID NO: 42, b is an integer of 15 to 1734, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 42, and where b is greater than or equal to a+14.

[0337] Features of Protein Encoded by Gene No: 33

[0338] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: MPRPSPLSSPGSPVTSQLCSPMPSLNPALPWGLLLALP GLSLHTPFQTLTAASPHQPSGDSAAHLSAHSFLLDSH (SEQ ID NO: 205); VPCGTACSVGAAA (SEQ ID NO: 206); and/or TSRSMF TSRPRTPWTSCLQIAP LALLQSLGIWQHSIGA (SEQ ID NO: 207). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0339] This gene is expressed primarily in neutrophils.

[0340] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune-related diseases and/or disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0341] The tissue distribution in neutrophils suggests that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis, treatment, and/or prevention of immune related diseases. Moreover, the expression of this gene product in neutrophils suggests a role in regulating the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses).

[0342] Since the gene is expressed in cells of lymphoid origin, the natural gene or protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0343] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 43 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 503 of SEQ ID NO: 43, b is an integer of 15 to 517, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 43, and where b is greater than or equal to a+14.

[0344] Features of Protein Encoded by Gene No: 34

[0345] This gene is expressed primarily in 12 week-old human embryos.

[0346] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, developmental abnormalities; cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the embryo, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types or cell types (e.g., embryonic, cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0347] The tissue distribution in embryonic tissue suggests that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis, detection, and/or treatment of developmental disorders. The relatively specific expression of this gene product during embryogenesis suggests that it may be a key player in the proliferation, maintenance, and/or differentiation of various cell types during development. It may also act as a morphogen to control cell and tissue type specification. Expression within embryonic tissue and other cellular sources marked by proliferating cells suggests that this protein may play a role in the regulation of cellular division. Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers.

[0348] Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and would be useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0349] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 44 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 472 of SEQ ID NO: 44, b is an integer of 15 to 486, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 44, and where b is greater than or equal to a+14.

[0350] Features of Protein Encoded by Gene No: 35

[0351] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: GTAGFSDLLLVNVMCQTRRSITFKNKLQKESRIYP (SEQ ID NO: 208). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0352] This gene is expressed primarily in CD34 depleted buffy coat (cord blood).

[0353] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, blood and immune diseases and/or disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, amniotic fluid, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0354] The tissue distribution in CD34 depleted buffy coat cells suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment of haemopoietic and immune disorders. Expression of this gene product in CD34 depleted buffy coat (cord blood) suggests a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses).

[0355] Since the gene is expressed in cells of lymphoid origin, the natural gene or protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0356] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 45 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 812 of SEQ ID NO: 45, b is an integer of 15 to 826, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 45, and where b is greater than or equal to a+14.

[0357] Features of Protein Encoded by Gene No: 36

[0358] This gene is expressed primarily in adipose tissue, and to a lesser extent, in placental tissue.

[0359] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, metabolic or reproductive disorders, particularly obesity. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the metabolic system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types or cell types (e.g., adipose, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0360] The tissue distribution in adipose tissue suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment of obesity by targeting adipose cells. Furthermore, the protein product of this clone may show utility in ameliorating conditions which occur secondary to aberrant fatty-acid metabolism (e.g., aberrant myelin sheath development), either directly or indirectly. Protein, as well as, antibodies directed against the protein may show utility as a tissue-specific marker and/or immunotherapy target for the above listed tissues.

[0361] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 46 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 680 of SEQ ID NO: 46, b is an integer of 15 to 694, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 46, and where b is greater than or equal to a+14.

[0362] Features of Protein Encoded by Gene No: 37

[0363] This gene is expressed primarily in adult pulmonary tissue.

[0364] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, pulmonary disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the pulmonary system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types or cell types (e.g., lung, cancerous and wounded tissues) or bodily fluids (e.g., lymph, pulmonary surfactant or sputum, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0365] The tissue distribution in pulmonary tissue suggests that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and/or treatment of certain pulmonary disorders, such as lung cancer, ARDS, and emphysema. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0366] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 47 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 842 of SEQ ID NO: 47, b is an integer of 15 to 856, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 47, and where b is greater than or equal to a+14.

[0367] Features of Protein Encoded by Gene No: 38

[0368] This gene is expressed primarily in amygdala tissue of the brain, and to a lesser extent, in infant brain tissues.

[0369] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, developmental and neurodegenerative diseases of the brain and nervous system. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the central nervous system (CNS), expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types or cell types (e.g., brain, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0370] The tissue distribution in neural tissues suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and/or diagnosis of behavioral or nervous system disorders, such as depression, schizophrenia, Alzheimer's disease, dementia, paranoia, autism, and addictive behavior. The amygdala processes sensory information and relays this to other areas of the brain including the endocrine and autonomic domains of the hypothalamus and the brain stem. Thus, the translation product of this gene may also be useful for the detection and/or treatment of neural disorders that impact processes mediated by the amygdala. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0371] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 48 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1629 of SEQ ID NO: 48, b is an integer of 15 to 1643, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 48, and where b is greater than or equal to a+14.

[0372] Features of Protein Encoded by Gene No: 39

[0373] This gene is expressed primarily in synovial hypoxia tissue.

[0374] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, connective tissue disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the connective tissue system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., skeletal, connective, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0375] The tissue distribution in synovial hypoxia tissue suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and/or diagnosis of diseases of connective tissue, particularly synovia, including but not limited to inflammation, rheumatoid arthritis, osteoarthritis, and cartilage tears and physical injury, as well as osteoporosis, tendonitis, chrondomalacia and inflammation. Furthermore, the translation product of this gene may be useful in the diagnosis and/or treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0376] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 49 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 695 of SEQ ID NO: 49, b is an integer of 15 to 709, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 49, and where b is greater than or equal to a+14.

[0377] Features of Protein Encoded by Gene No: 40

[0378] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: PFRNSRVRPKGSRDALSWSSCTGPQPGTSATVGSLLCGG VPCIAGHPAASPASCSVPVAPHPAVVTAQVSRCAECPLVMLRGTGVLPPGFE RCLTPTSGVSLPCV (SEQ ID NO: 209). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0379] This gene is expressed primarily in synovial cells stimulated with IL-1 and TNF.

[0380] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, inflammation of connective tissue. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of connective tissue, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types or cell types (e.g., connective, skeletal, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0381] The tissue distribution in synovial cells suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and/or diagnosis of inflammation of connective tissues, particularly the synovium, in diseases such as rheumatoid arthritis, sepsis, infection of the joint, and tissue damage from physical injury. Furthermore, the expression of this gene product may be useful in the diagnosis or treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0382] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 50 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 527 of SEQ ID NO: 50, b is an integer of 15 to 541, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 50, and where b is greater than or equal to a+14.

[0383] Features of Protein Encoded by Gene No: 41

[0384] When tested against U937 myeloid cell lines as well as Jurkat T-cell cell lines, supernatants removed from cells containing this gene activated the GAS assay. Therefore, it is likely that this gene activates myeloid cells, and to a lesser extent, in immune and hematopoietic cells through the Jak-STAT signal transduction pathway. Gamma activating sequence (GAS) is a promoter element found upstream of many genes involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.

[0385] This gene is expressed primarily in melanocytes, and to a lesser extent, in the synovium, testes, and CD34 cells.

[0386] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, musculo-skeletal diseases and disorders, such as skin discoloration or arthritis, as well as male reproductive disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the musculo-skeletal and reproductive systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types or cell types (e.g., musculo-skeletal, reproductive, bone, cartilage, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0387] The tissue distribution in synovium suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and/or diagnosis of diseases affecting the skeletal system, in particular osteoporosis, as well as disorders afflicting connective tissues (e.g., arthritis, trauma, tendonitis, chrondomalacia and inflammation), such as in the diagnosis or treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid).

[0388] Alternatively, the tissue distribution in testes tissue indicates that the protein product of this clone is useful for the treatment and diagnosis of conditions concerning proper testicular function (e.g., endocrine function, sperm maturation), as well as cancer. Therefore, this gene product is useful in the treatment of male infertility and/or impotence. This gene product is also useful in assays designed to identify binding agents, as such agents (antagonists) are useful as male contraceptive agents. Similarly, the protein is believed to be useful in the treatment and/or diagnosis of testicular cancer. The testes are also a site of active gene expression of transcripts that may be expressed, particularly at low levels, in other tissues of the body. Therefore, this gene product may be expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0389] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 51 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 706 of SEQ ID NO: 51, b is an integer of 15 to 720, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 51, and where b is greater than or equal to a+14.

[0390] Features of Protein Encoded by Gene No: 42

[0391] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: MDTYTFLIKICKIFCSFLKCHIQVCGHLLFLIFTSIKWARK QHHCSRCKAIGLSS (SEQ ID NO: 210). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0392] This gene is expressed primarily in synovial tissue and neutrophils.

[0393] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, musculo-skeletal and immune diseases. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and musculo-skeletal systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types or cell types (e.g., immune, skeletal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0394] The tissue distribution in synovium and neutrophils suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and/or diagnosis of immune diseases, particularly inflammatory conditions such as arthritis. Expression of this gene product in neutrophils suggests a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses).

[0395] Since the gene is expressed in cells of lymphoid origin, the gene or protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. Furthermore, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.

[0396] In addition, the expression of this gene product in synovium suggests a role in the detection and treatment of disorders and conditions affecting the skeletal system, in particular osteoporosis as well as disorders afflicting connective tissues (e.g., arthritis, trauma, tendonitis, chrondomalacia and inflammation), such as in the diagnosis or treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familial arthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0397] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 52 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 965 of SEQ ID NO: 52, b is an integer of 15 to 979, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 52, and where b is greater than or equal to a+14.

[0398] Features of Protein Encoded by Gene No: 43

[0399] This gene is expressed primarily in CD34 depleted buffy coat (cord blood).

[0400] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, blood, developmental, and immune disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0401] The tissue distribution in CD34 depleted buffy coat suggests that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and/or treatment of haemopoietic and immune disorders. Furthermore, expression of this gene product in CD34 depleted buffy coat (cord blood) suggests a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses).

[0402] Since the gene is expressed in cells of lymphoid origin, the gene or protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0403] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 53 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 366 of SEQ ID NO: 53, b is an integer of 15 to 380, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 53, and where b is greater than or equal to a+14.

[0404] Features of Protein Encoded by Gene No: 44

[0405] The translation product of this gene shares sequence homology with the conserved NADH-isocitrate dehydrogenase which is thought to be important in the proliferation of lymphocytes. When tested against Jurkat T-cell cell lines, supernatants removed from cells containing this gene activated the GAS assay. Therefore, it is likely that this gene activates T-cells, and to a lesser extent in immune and hematopoietic cells and tissues, through the Jak-STAT signal transduction pathway. Gamma activating sequence (GAS) is a promoter element found upstream of many genes involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.

[0406] In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequences:

[0407] DPRLAVLLLGVQILVERWRLQWDHYYLCPHRVQAEED VEKSQWNYPEHPGGDCLPGAHHLQKHPTPSPWLDQAHEIWQARPWXPVQG HRLCGRPGRHFQNGLHPKRWQWCQGVGSVQLPRSGVGMGMYNTDESISGF AHSCFQYAIQKKWPLYMSTKNTILKAYDGRFKDIFQEIFDKHYKTDFDKNKI WYEHRLIDDMVAQVLKSSGGFVWACKNYDGDVQSDILAQGFGSLGLMTSV LVCPDGKTIEAEAAHGTVTRHYREHQKGRPTSTNPIASIFAWTRGLEHRGKLD GNQDLIRFAQMLEKVCVETVESGAMTKDLAGCIHGLSNVKLNEHFLNTTDFL DTIKSNLDRALGRQ (SEQ ID NO: 21 1); DPRLAVLLLGVQILVERWRLQWDHY YLCPHRVQAEEDVEKSQWNYPEH (SEQ ID NO: 212); PGGDCLPGAHHLQKH PTPSPWLDQAHHHWQARPWXPVQGHRLCGRPGRHFQNGL (SEQ ID NO: 213); HPKRWQWCQGVGSVQLPRSGVGMGMYN ESISGFAHSCFQYA IQKKWPLYMSTKNTILKAYDGRFKDIF (SEQ ID NO: 214); QEIFDKHYKTDF DKNKIWYEHRLIDDMVAQVLKSSGGFVWACKNYDGDVQSDILAQGFGSLGL MTSVLVC (SEQ ID NO: 215); PDGKTIEAEAAHGTVTRHYREHQKGRPTSTNP IASIFAWTRGLEHRGKLDGNQDLIRFAQMLEKVCVETV (SEQ ID NO: 216); and/or ESGAMTKDLAGCIHGLSNVKLNEHFLNTTDFLDTIKSNLDRALGRQ (SEQ ID NO: 217). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0408] This gene is expressed primarily in T-cells and B-cells, and to a lesser extent in breast cancer tissue.

[0409] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, breast cancer, leukemia, HIV, and tonsillitis. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune/lymphoid system, and breast cancers, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, reproductive, breast, cancerous and wounded tissues) or bodily fluids (e.g., lymph, breast milk, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0410] The tissue distribution in immune cells, combined with the detected gas biological activity, and its homology to the NADH-isocitrate dehydrogenase, suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and/or diagnosis of diseases related to the proliferation of lymphoid cells. Furthermore, it could be especially useful in helping the body to produce mature lymphocytes to enhance the immune system. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0411] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 54 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2009 of SEQ ID NO: 54, b is an integer of 15 to 2023, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 54, and where b is greater than or equal to a+14.

[0412] Features of Protein Encoded by Gene No: 45

[0413] This gene is expressed primarily in PHA treated T-cells.

[0414] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, T-cell disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0415] The tissue distribution in T-cells suggests that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and/or treatment of immune disorders involving T-cells. Furthermore, the gene product may play a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses).

[0416] Since the gene is expressed in cells of lymphoid origin, the gene or protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Expression of this gene product in T cells also strongly suggests a role for this protein in immune function and immune surveillance. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0417] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 55 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 871 of SEQ ID NO: 55, b is an integer of 15 to 885, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 55, and where b is greater than or equal to a+14.

[0418] Features of Protein Encoded by Gene No: 46

[0419] This gene is expressed primarily in fetal heart tissue.

[0420] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, mitrovalve prolapse, congenital heart diseases or arythmic heartbeats. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the cardiovascular system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types or cell types (e.g., cardiac, developmental, cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0421] The tissue distribution in fetal heart suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and/or diagnosis of heart diseases especially in the developing fetus. Furthermore, the tissue distribution in fetal heart tissue indicates that the protein product of this gene is useful for the diagnosis, treatment, and/or prevention of conditions and pathologies of the cardiovascular system, such as heart disease, restenosis, atherosclerosis, stoke, angina, thrombosis, and wound healing. The sequence itself could be used in genetic therapy, in utero, to correct defects in the developing fetus. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0422] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 56 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1092 of SEQ ID NO: 56, b is an integer of 15 to 1106, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 56, and where b is greater than or equal to a+14.

[0423] Features of Protein Encoded by Gene No: 47

[0424] This gene is expressed primarily in spleen tissue of chronic lymphocytic leukemia patient.

[0425] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, leukemia. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0426] The tissue distribution in spleen tissue suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and/or diagnosis of patients with leukemia. Expression of this gene product in spleen tissue suggests a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses).

[0427] Since the gene is expressed in cells of lymphoid origin, the gene or protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0428] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 57 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 750 of SEQ ID NO: 57, b is an integer of 15 to 764, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 57, and where b is greater than or equal to a+14.

[0429] Features of Protein Encoded by Gene No: 48

[0430] This gene is expressed primarily in liver cancer tissue, and to a lesser extent in bone marrow, neutrophils, and CD34 cells.

[0431] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, liver cancer and scerosis of the liver. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the hepatic and lymphatic systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., liver, immune, hematopoiectic, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0432] The tissue distribution in cancerous liver tissue suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and/or diagnosis of liver cancer, possibly before the onset of symptoms. Similarly, this gene would be useful for the detection and treatment of liver disorders and cancers (e.g., hepatoblastoma, jaundice, hepatitis, liver metabolic diseases and conditions that are attributable to the differentiation of hepatocyte progenitor cells). Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0433] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 58 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 724 of SEQ ID NO: 58, b is an integer of 15 to 738, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 58, and where b is greater than or equal to a+14.

[0434] Features of Protein Encoded by Gene No: 49

[0435] This gene is expressed primarily in synovial hypoxia tissue.

[0436] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, connective tissue and joint disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of connective tissue, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., connective, skeletal, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0437] The tissue distribution in synovial hypoxia tissue suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and/or diagnosis of diseases of connective tissue, particularly synovia, including but not limited to inflammation, rheumatoid arthritis, osteoarthritis, and cartilage tears and physical injury. Furthermore, polynucleotides and polypeptides corresponding to this gene are useful in the diagnosis or treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, farmilial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0438] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 59 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 427 of SEQ ID NO: 59, b is an integer of 15 to 441, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 59, and where b is greater than or equal to a+14.

[0439] Features of Protein Encoded by Gene No: 50

[0440] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: MIMGYKSQKTFGLFDLXXVKGKTSVLEFDFWVQIPVA SLLALWLNRLLNSVKWALKXCVIHSVAVNX (SEQ ID NO: 218). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0441] This gene is expressed primarily in B-cell lymphoma tissue.

[0442] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, B-cell lymphoma. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0443] The tissue distribution in B-cell lymphoma suggests that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and/or treatment of immune or hematopoietic disorders, particularly those involving proliferative cells or tissues such as in cancers. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0444] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 60 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 770 of SEQ ID NO: 60, b is an integer of 15 to 784, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 60, and where b is greater than or equal to a+14.

[0445] Features of Protein Encoded by Gene No: 51

[0446] This gene is expressed primarily in synovial hypoxia tissue.

[0447] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, connective tissue disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of connective tissue, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., connective, skeletal, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0448] The tissue distribution in synovial hypoxia tissue suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and/or diagnosis of diseases of connective tissue, particularly synovia, including but not limited to inflammation, rheumatoid arthritis, osteoarthritis, and cartilage tears and physical injury. Furthermore, polynucleotides and polypeptides corresponding to this gene are useful in the diagnosis or treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, joint abnormalities, and chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0449] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 61 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 526 of SEQ ID NO: 61, b is an integer of 15 to 540, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 61, and where b is greater than or equal to a+14.

[0450] Features of Protein Encoded by Gene No: 52

[0451] This gene is expressed primarily in synovial cells stimulated with IL-1 and TNF, and also in neutrophils.

[0452] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, inflammation of connective tissue and joints. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of connective tissue, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., connective, skeletal, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0453] The tissue distribution in synovial cells suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and/or diagnosis of inflammation of connective tissues, particularly the synovium, in diseases such as rheumatoid arthritis, sepsis, infection of the joint, and tissue damage from physical injury. Furthermore, polynucleotides and polypeptides corresponding to this gene are useful in the diagnosis or treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, joint abnormalities, and chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0454] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 62 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 590 of SEQ ID NO: 62, b is an integer of 15 to 604, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 62, and where b is greater than or equal to a+14.

[0455] Features of Protein Encoded by Gene No: 53

[0456] In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: MKSFPSTYFKSSSFQNTKYQTGVISVLISYEIEYAAFYHL SCKITLPSSVSRNCFISEXLVASQCLDT (SEQ ID NO: 219). Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0457] This gene is expressed primarily in frontal cortex tissue sampled from an epileptic patient.

[0458] Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, neural disorders, such as epilepsy. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the nervous system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., brain, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

[0459] The tissue distribution in frontal cortex tissue suggests that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and/or diagnosis of epilepsy and related brain disorders. Elevated expression of this gene product within the frontal cortex tissue of the brain suggests that it may be involved in neuronal survival; synapse formation; conductance; neural differentiation, etc. Such involvement may impact many processes, such as learning and cognition. It may also be useful in the treatment of such neurodegenerative disorders as schizophrenia; ALS; or Alzheimer's. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

[0460] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 63 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 738 of SEQ ID NO: 63, b is an integer of 15 to 752, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 63, and where b is greater than or equal to a+14. TABLE 1A 5′ NT of First Last ATCC NT 5′ NT 3′ NT 5′ NT First AA AA AA First Last Deposit SEQ Total of of of AA of SEQ of of AA of AA cDNA NO: Z ID NT Clone Clone Start Signal ID Sig Sig Secreted of Gene No. Clone ID and Date Vector NO: X Seq. Seq. Seq. Codon Pep NO: Y Pep Pep Portion ORF 1 HSIDU19 209244 Uni-ZAP XR 11 680 1 680 352 352 67 1 21 22 73 Sep. 12, 1997 2 HPRSB76 209244 pBluescript 12 741 1 741 127 127 68 1 22 23 59 Sep. 12, 1997 3 HTEIL66 209244 Uni-ZAP XR 13 619 1 619 123 123 69 1 24 25 134 Sep. 12, 1997 4 HSNAY92 209244 Uni-ZAP XR 14 611 1 611 127 127 70 1 29 30 35 Sep. 12, 1997 5 HSABG21 209244 pBluescript 15 585 1 585 96 96 71 1 24 25 125 Sep. 12, 1997 SK- 6 HSAXB32 209244 Uni-ZAP XR 16 1040 1 1040 97 97 72 1 36 37 51 Sep. 12, 1997 7 HPEAD48 209244 Uni-ZAP XR 17 625 1 625 203 203 73 1 18 19 97 Sep. 12, 1997 8 HPVAB94 209244 Uni-ZAP XR 18 819 1 819 80 80 74 1 25 26 44 Sep. 12, 1997 9 HSAXB81 209244 Uni-ZAP XR 19 782 1 782 143 143 75 1 20 21 47 Sep. 12, 1997 10 HSAYC21 209244 Uni-ZAP XR 20 655 1 655 155 155 76 1 26 Sep. 12, 1997 11 HSLCU73 209244 Uni-ZAP XR 21 798 1 798 7 7 77 1 22 23 41 Sep. 12, 1997 12 HSSFZ70 209244 Uni-ZAP XR 22 646 1 646 212 212 78 1 22 23 22 Sep. 12, 1997 13 HTEIP36 209244 Uni-ZAP XR 23 752 1 752 22 22 79 1 19 20 58 Sep. 12, 1997 14 HYBAY77 209244 Uni-ZAP XR 24 815 60 815 157 157 80 1 44 45 47 Sep. 12, 1997 15 HROAE78 209244 Uni-ZAP XR 25 878 1 878 132 132 81 1 16 17 52 Sep. 12, 1997 16 HSAVP17 209244 Uni-ZAP XR 26 850 1 850 69 69 82 1 18 19 44 Sep. 12, 1997 17 HSIEA14 209244 Uni-ZAP XR 27 788 1 788 141 141 83 1 22 23 60 Sep. 12, 1997 18 HSNAQ47 209244 Uni-ZAP XR 28 838 1 838 80 80 84 1 21 22 253 Sep. 12, 1997 18 HSNAQ47 209244 Uni-ZAP XR 64 848 1 848 85 85 120 1 21 22 24 Sep. 12, 1997 19 HODDN65 209244 Uni-ZAP XR 29 755 1 755 251 251 85 1 14 15 20 Sep. 12, 1997 20 HPEAD79 209244 Uni-ZAP XR 30 813 1 813 51 51 86 1 15 16 41 Sep. 12, 1997 21 HRDED19 209244 Uni-ZAP XR 31 513 1 513 75 75 87 1 20 21 47 Sep. 12, 1997 22 HSAYS89 209244 Uni-ZAP XR 32 576 1 576 94 94 88 1 15 16 43 Sep. 12, 1997 23 HTODK73 209244 Uni-ZAP XR 33 1019 4 1019 43 43 89 1 23 24 59 Sep. 12, 1997 24 HSVAM10 209244 Uni-ZAP XR 34 433 1 433 46 46 90 1 27 28 51 Sep. 12, 1997 25 HSNBN57 209244 Uni-ZAP XR 35 642 1 642 198 198 91 1 20 21 31 Sep. 12, 1997 26 HSVBD22 209244 Uni-ZAP XR 36 667 1 667 61 61 92 1 24 25 35 Sep. 12, 1997 27 HSAWA27 209244 Uni-ZAP XR 37 654 1 654 319 319 93 1 29 30 49 Sep. 12, 1997 28 HSFAH43 209244 Uni-ZAP XR 38 731 1 731 191 191 94 1 22 23 24 Sep. 12, 1997 29 HSPAA60 209244 pSport1 39 378 1 378 198 198 95 1 45 46 46 Sep. 12, 1997 30 HFAEF57 209277 Uni-ZAP XR 40 642 1 642 232 232 96 1 42 43 86 Sep. 18, 1997 31 HEGAH43 209277 Uni-ZAP XR 41 442 1 442 29 29 97 1 20 21 111 Sep. 18, 1997 32 HAGDG59 209277 Uni-ZAP XR 42 1734 44 1717 124 124 98 1 18 19 300 Sep. 18, 1997 33 HNGBX63 209277 Uni-ZAP XR 43 517 1 517 120 120 99 1 15 16 104 Sep. 18, 1997 34 HE2AG50 209277 Uni-ZAP XR 44 486 1 486 19 19 100 1 32 33 43 Sep. 18, 1997 35 HCUIN80 209277 ZAP Express 45 826 1 826 106 106 101 1 16 17 49 Sep. 18, 1997 36 HADCL29 209277 pSport1 46 694 1 694 248 248 102 1 16 17 47 Sep. 18, 1997 37 HAPPS89 209277 Uni-ZAP XR 47 856 1 856 54 54 103 1 29 30 99 Sep. 18, 1997 38 HFGAH44 209277 Uni-ZAP XR 48 1643 1 1643 34 34 104 1 20 21 58 Sep. 18, 1997 39 HFIHZ96 209277 pSport1 49 709 1 709 39 39 105 1 24 25 64 Sep. 18, 1997 40 HFIUR10 209277 pSport1 50 541 1 541 50 50 106 1 22 23 44 Sep. 18, 1997 41 HLDNA86 209277 pCMVSport 51 720 1 717 45 45 107 1 31 32 92 Sep. 18, 1997 3.0 42 HNGAN75 209277 Uni-ZAP XR 52 979 1 979 41 41 108 1 25 26 25 Sep. 18, 1997 43 HCUIO20 209277 ZAP Express 53 380 1 380 43 43 109 1 19 20 67 Sep. 18, 1997 44 HLTEF12 209277 Uni-ZAP XR 54 2023 624 1498 686 686 110 1 21 22 44 Sep. 18, 1997 45 HCFBJ91 209277 pSport1 55 885 1 885 61 61 111 1 20 21 52 Sep. 18, 1997 46 HHFHP90 209277 Uni-ZAP XR 56 1106 1 1106 42 42 112 1 14 15 43 Sep. 18, 1997 47 HLYCQ48 209277 pSport1 57 764 1 764 58 58 113 1 40 41 64 Sep. 18, 1997 48 HHLAB07 209277 pBluescript 58 738 1 738 108 108 114 1 34 35 69 Sep. 18, 1997 SK- 49 HFOXE30 209277 pSport1 59 441 1 441 38 38 115 1 18 19 53 Sep. 18, 1997 50 HBJEL68 209277 Uni-ZAP XR 60 784 1 784 109 109 116 1 33 34 41 Sep. 18, 1997 50 HBJEL68 209277 Uni-ZAP XR 65 769 1 769 111 111 121 1 26 Sep. 18, 1997 51 HFOXA73 209277 pSport1 61 540 1 540 25 25 117 1 17 18 52 Sep. 18, 1997 51 HFOXA73 209277 pSport1 66 539 1 539 15 15 122 1 17 Sep. 18, 1997 52 HFIUR35 209277 pSport1 62 604 1 604 42 42 118 1 31 32 70 Sep. 18, 1997 53 HFPDE86 209277 Uni-ZAP XR 63 752 1 752 300 300 119 1 16 Sep. 18, 1997

[0461] TABLE 1B AA Tissue Distribution SEQ Library code: count OMIM Gene cDNA Clone Contig SEQ ID ORF ID (see Table IV for Cytologic Disease No: ID ID: NO: X (From-To) NO: Y Predicted Epitopes Library Codes) Band Reference(s) 1 HSIDU19 520372 11 352-573 67 Thr-27 to Arg-45. AR251: 6, AR310: 6, 6p22- 106300, AR186: 6, AR249: 5, p21.3 108800, AR263: 5, AR052: 5, 120290, AR265: 5, AR253: 5, 120290, AR292: 4, AR055: 4, 120810, AR061: 4, AR224: 4, 120820, AR247: 3, AR194: 3, 142857, AR309: 3, AR169: 3, 142858, AR225: 3, AR266: 3, 150270, AR312: 3, AR202: 3, 167250, AR215: 3, AR033: 3, 170261, AR163: 3, AR053: 3, 177900, AR204: 3, AR283: 3, 179450, AR162: 3, AR295: 3, 201910, AR259: 3, AR198: 2, 217000, AR183: 2, AR277: 2, 222100, AR282: 2, AR164: 2, 233100, AR226: 2, AR233: 2, 235200, AR245: 2, AR300: 2, 248611, AR299: 2, AR311: 2, 256550, AR193: 2, AR221: 2, 256550, AR096: 2, AR184: 2, 600202, AR217: 2, AR177: 2, 600261, AR237: 2, AR223: 2, 601868, AR231: 2, AR089: 2, 602280, AR232: 2, AR238: 2, 602475 AR273: 2, AR197: 2, AR229: 2, AR192: 2, AR175: 2, AR291: 2, AR185: 2, AR294: 2, AR264: 2, AR293: 2, AR313: 2, AR161: 2, AR284: 2, AR060: 2, AR227: 2, AR213: 2, AR214: 2, AR165: 1, AR269: 1, AR205: 1, AR271: 1, AR216: 1, AR267: 1, AR201: 1, AR243: 1, AR234: 1, AR182: 1, AR218: 1, AR222: 1, AR256: 1, AR298: 1, AR316: 1, AR039: 1, AR255: 1, AR258: 1, AR270: 1, AR219: 1 H0036: 1 2 HPRSB76 526310 12 127-306 68 AR169: 5, AR282: 4, 15q11- 103581, AR253: 4, AR266: 4, q13 146150, AR221: 3, AR198: 2, 176270, AR245: 2, AR295: 2, 218000, AR272: 2, AR285: 2, 227220, AR176: 2, AR225: 2, 601623, AR286: 2, AR289: 2, 601800, AR300: 2, AR214: 1, 601889, AR287: 1, AR055: 1, 602117 AR182: 1, AR199: 1, AR212: 1, AR269: 1, AR170: 1, AR178: 1, AR297: 1, AR161: 1, AR293: 1, AR162: 1 H0211: 1 and L0759: 1. 3 HTEIL66 520125 13 123-527 69 Glu-32 to Asn-54, AR161: 7, AR162: 7, His-98 to Arg-106, AR176: 7, AR163: 7, Ser-126 to Ser-134. AR266: 6, AR181: 6, AR229: 6, AR180: 6, AR182: 6, AR060: 6, AR228: 6, AR183: 6, AR173: 5, AR165: 5, AR261: 5, AR309: 5, AR269: 5, AR164: 5, AR239: 5, AR257: 5, AR313: 5, AR233: 5, AR289: 5, AR166: 5, AR236: 4, AR275: 4, AR268: 4, AR300: 4, AR240: 4, AR255: 4, AR235: 4, AR277: 4, AR274: 4, AR267: 4, AR264: 4, AR177: 4, AR247: 4, AR238: 4, AR270: 4, AR217: 4, AR170: 4, AR214: 4, AR179: 4, AR089: 4, AR104: 4, AR175: 4, AR061: 4, AR224: 4, AR237: 4, AR283: 3, AR282: 3, AR291: 3, AR316: 3, AR096: 3, AR293: 3, AR299: 3, AR221: 3, AR196: 3, AR193: 3, AR296: 3, AR272: 3, AR262: 3, AR174: 3, AR295: 3, AR185: 3, AR231: 3, AR222: 3, AR178: 3, AR226: 3, AR230: 3, AR290: 3, AR312: 3, AR234: 3, AR227: 3, AR204: 3, AR053: 3, AR288: 3, AR191: 3, AR201: 3, AR258: 3, AR168: 3, AR311: 3, AR297: 3, AR215: 3, AR055: 3, AR246: 3, AR285: 2, AR287: 2, AR286: 2, AR294: 2, AR263: 2, AR232: 2, AR171: 2, AR250: 2, AR216: 2, AR243: 2, AR200: 2, AR203: 2, AR033: 2, AR190: 2, AR225: 2, AR189: 2, AR260: 2, AR199: 2, AR211: 2, AR256: 2, AR188: 2, AR212: 2, AR210: 1, AR172: 1, AR218: 1, AR219: 1, AR198: 1, AR252: 1 L0794: 7, L0758: 5 and H0038: 1. 4 HSNAY92 526730 14 127-234 70 AR176: 3, AR282: 2, AR223: 2, AR204: 2, AR039: 2, AR221: 2, AR169: 2, AR053: 2, AR195: 2, AR205: 2, AR309: 2, AR264: 2, AR181: 2, AR291: 1, AR262: 1, AR270: 1, AR216: 1, AR183: 1, AR201: 1, AR269: 1, AR240: 1, AR224: 1, AR294: 1, AR179: 1, AR247: 1, AR198: 1 5 HSABG21 526366 15  96-473 71 Thr-32 to Leu-43. AR250: 8, AR171: 4, AR225: 4, AR263: 4, AR060: 3, AR282: 3, AR235: 2, AR221: 2, AR240: 2, AR261: 2, AR168: 2, AR283: 2, AR223: 2, AR178: 2, AR257: 1, AR104: 1, AR089: 1, AR170: 1, AR172: 1, AR185: 1, AR217: 1, AR277: 1, AR162: 1, AR214: 1, AR163: 1, AR199: 1, AR316: 1, AR096: 1, AR272: 1 T0039: 1 and L0751: 1. 6 HSAXB32 520470 16 97-252 72 AR313: 48, AR089: 39, AR299: 30, AR316: 26, AR096: 24, AR104: 20, AR185: 19, AR060: 17, AR277: 16, AR240: 11, AR161: 10, AR162: 10, AR163: 10, AR165: 9, AR164: 9, AR166: 8, AR275: 7, AR196: 6, AR283: 6, AR282: 6, AR053: 5, AR274: 5, AR312: 5, AR300: 4, AR173: 4, AR175: 4, AR257: 4, AR264: 4, AR211: 4, AR308: 4, AR309: 4, AR247: 4, AR174: 4, AR195: 4, AR213: 4, AR262: 3, AR178: 3, AR191: 3, AR236: 3, AR199: 3, AR189: 3, AR203: 3, AR200: 3, AR258: 3, AR261: 3, AR229: 3, AR286: 3, AR197: 3, AR297: 3, AR218: 3, AR242: 2, AR212: 2, AR233: 2, AR285: 2, AR295: 2, AR179: 2, AR255: 2, AR234: 2, AR230: 2, AR238: 2, AR288: 2, AR201: 2, AR243: 2, AR287: 2, AR176: 2, AR294: 2, AR177: 2, AR181: 2, AR204: 2, AR296: 2, AR215: 2, AR183: 2, AR182: 2, AR293: 2, AR266: 2, AR033: 2, AR272: 2, AR223: 2, AR225: 2, AR219: 2, AR217: 2, AR226: 2, AR237: 2, AR231: 2, AR269: 2, AR172: 2, AR228: 2, AR260: 2, AR235: 2, AR311: 1, AR193: 1, AR268: 1, AR239: 1, AR188: 1, AR227: 1, AR291: 1, AR289: 1, AR290: 1, AR216: 1, AR267: 1 S0114: 1 and L0608: 1. 7 HPEAD48 520367 17 203-496 73 Gln-51 to Thr-61, AR196: 9, AR161: 9, Ser-65 to Thr-71, AR162: 9, AR163: 8, Pro-85 to Gln-91. AR173: 8, AR169: 8, AR171: 8, AR313: 7, AR168: 7, AR223: 7, AR175: 6, AR263: 6, AR240: 6, AR096: 6, AR258: 6, AR180: 5, AR264: 5, AR261: 5, AR262: 5, AR257: 5, AR229: 5, AR176: 5, AR165: 5, AR300: 5, AR282: 5, AR164: 5, AR214: 5, AR269: 5, AR185: 5, AR275: 5, AR089: 5, AR166: 5, AR274: 4, AR270: 4, AR174: 4, AR199: 4, AR181: 4, AR217: 4, AR179: 4, AR191: 4, AR253: 4, AR247: 4, AR183: 4, AR234: 4, AR170: 4, AR266: 4, AR177: 4, AR299: 4, AR218: 4, AR236: 4, AR309: 4, AR312: 4, AR316: 4, AR238: 4, AR233: 4, AR235: 4, AR189: 4, AR215: 4, AR213: 4, AR311: 4, AR225: 3, AR224: 3, AR277: 3, AR293: 3, AR200: 3, AR212: 3, AR104: 3, AR033: 3, AR178: 3, AR226: 3, AR255: 3, AR230: 3, AR296: 3, AR268: 3, AR060: 3, AR203: 3, AR291: 3, AR188: 3, AR237: 3, AR172: 3, AR182: 3, AR285: 3, AR283: 3, AR198: 3, AR216: 2, AR192: 2, AR219: 2, AR228: 2, AR227: 2, AR295: 2, AR287: 2, AR267: 2, AR286: 2, AR260: 2, AR294: 2, AR239: 2, AR231: 2, AR297: 2, AR201: 2, AR290: 2, AR222: 2, AR053: 2, AR272: 2, AR190: 2, AR289: 2, AR288: 2, AR256: 2, AR204: 2, AR232: 2, AR207: 1, AR210: 1, AR055: 1, AR197: 1, AR193: 1, AR308: 1, AR205: 1 H0165: 1 8 HPVAB94 526749 18  80-214 74 AR192: 5, AR242: 3, AR214: 3, AR195: 2, AR264: 2, AR168: 2, AR225: 2, AR277: 2, AR257: 1, AR172: 1, AR282: 1, AR171: 1, AR255: 1, AR275: 1, AR270: 1, AR296: 1, AR165: 1, AR182: 1, AR224: 1, AR295: 1 S0013: 1 9 HSAXB81 520471 19 143-286 75 AR313: 38, AR242: 24, AR300: 23, AR173: 23, AR204: 23, AR178: 22, AR180: 21, AR089: 21, AR163: 21, AR162: 21, AR229: 21, AR247: 21, AR164: 21, AR166: 21, AR165: 20, AR161: 20, AR096: 20, AR175: 19, AR299: 18, AR258: 17, AR293: 17, AR197: 17, AR269: 16, AR179: 16, AR270: 16, AR183: 16, AR176: 16, AR262: 16, AR257: 15, AR182: 15, AR218: 15, AR240: 15, AR181: 14, AR226: 14, AR296: 14, AR268: 14, AR174: 14, AR234: 13, AR233: 13, AR312: 13, AR185: 13, AR238: 13, AR193: 12, AR245: 12, AR316: 12, AR201: 12, AR297: 11, AR230: 11, AR264: 11, AR243: 11, AR231: 11, AR177: 11, AR294: 11, AR282: 11, AR192: 10, AR203: 10, AR237: 10, AR039: 10, AR196: 10, AR199: 10, AR236: 10, AR267: 9, AR285: 9, AR060: 9, AR053: 9, AR266: 9, AR239: 9, AR191: 9, AR033: 9, AR288: 9, AR198: 9, AR261: 9, AR228: 9, AR286: 8, AR287: 8, AR195: 8, AR205: 8, AR291: 8, AR255: 8, AR219: 8, AR274: 8, AR212: 8, AR277: 8, AR189: 7, AR253: 7, AR289: 7, AR252: 7, AR295: 7, AR250: 7, AR171: 7, AR213: 7, AR290: 7, AR260: 7, AR271: 7, AR275: 7, AR227: 7, AR104: 7, AR254: 7, AR256: 7, AR200: 6, AR207: 6, AR308: 6, AR309: 6, AR283: 6, AR172: 5, AR235: 5, AR232: 5, AR188: 5, AR061: 5, AR263: 5, AR169: 5, AR272: 5, AR168: 5, AR211: 5, AR055: 5, AR246: 4, AR190: 4, AR311: 3, AR170: 3, AR221: 2, AR210: 2, AR217: 2, AR222: 2, AR224: 2, AR225: 2, AR216: 2 S0114: 1 and H0542: 1. 10 HSAYC21 526188 20 155-235 76 AR313: 10, AR242: 8, AR269: 8, AR089: 8, AR161: 7, AR165: 7, AR229: 7, AR228: 7, AR162: 7, AR182: 7, AR164: 7, AR060: 7, AR163: 7, AR166: 7, AR096: 7, AR181: 7, AR233: 7, AR267: 6, AR299: 6, AR176: 6, AR238: 6, AR053: 6, AR239: 6, AR316: 6, AR196: 6, AR300: 6, AR257: 6, AR235: 5, AR236: 5, AR240: 5, AR268: 5, AR172: 5, AR177: 5, AR175: 5, AR173: 5, AR266: 5, AR309: 5, AR180: 5, AR191: 5, AR247: 5, AR237: 5, AR183: 5, AR179: 5, AR270: 5, AR226: 5, AR231: 5, AR104: 5, AR200: 4, AR168: 4, AR185: 4, AR262: 4, AR261: 4, AR255: 4, AR264: 4, AR204: 4, AR296: 4, AR253: 4, AR293: 4, AR282: 4, AR312: 4, AR192: 4, AR171: 4, AR258: 4, AR234: 4, AR189: 4, AR294: 4, AR230: 4, AR227: 4, AR190: 4, AR178: 4, AR055: 4, AR308: 4, AR285: 3, AR198: 3, AR212: 3, AR203: 3, AR174: 3, AR199: 3, AR291: 3, AR061: 3, AR277: 3, AR289: 3, AR287: 3, AR215: 3, AR193: 3, AR218: 3, AR207: 3, AR195: 3, AR290: 3, AR260: 3, AR272: 3, AR033: 3, AR283: 3, AR224: 3, AR201: 3, AR223: 3, AR288: 3, AR297: 3, AR286: 3, AR232: 3, AR243: 3, AR263: 2, AR225: 2, AR214: 2, AR256: 2, AR275: 2, AR219: 2, AR274: 2, AR188: 2, AR246: 2, AR295: 2, AR216: 2, AR210: 2, AR213: 2, AR217: 2, AR311: 2, AR271: 1, AR205: 1, AR211: 1, AR170: 1 S0114: 1, H0254: 1, H0637: 1 and L0783: 1. 11 HSLCU73 520237 21  7-132 77 AR173: 13, AR313: 13, AR229: 11, AR165: 10, AR242: 10, AR164: 10, AR178: 10, AR161: 10, AR162: 10, AR166: 10, AR163: 10, AR180: 9, AR300: 9, AR175: 8, AR192: 8, AR262: 8, AR181: 8, AR247: 8, AR257: 8, AR179: 8, AR233: 7, AR198: 7, AR197: 7, AR226: 7, AR204: 7, AR258: 7, AR176: 7, AR254: 7, AR201: 7, AR234: 7, AR177: 7, AR293: 6, AR193: 6, AR183: 6, AR039: 6, AR238: 6, AR182: 6, AR218: 6, AR174: 6, AR245: 6, AR266: 6, AR264: 6, AR270: 6, AR312: 5, AR228: 5, AR261: 5, AR053: 5, AR275: 5, AR207: 5, AR239: 5, AR269: 5, AR274: 5, AR240: 5, AR299: 5, AR271: 5, AR230: 5, AR237: 5, AR185: 5, AR089: 5, AR231: 5, AR268: 5, AR243: 5, AR096: 4, AR250: 4, AR282: 4, AR195: 4, AR199: 4, AR297: 4, AR267: 4, AR255: 4, AR277: 4, AR236: 4, AR033: 4, AR212: 4, AR286: 4, AR263: 4, AR227: 4, AR205: 4, AR252: 4, AR296: 4, AR246: 4, AR219: 4, AR309: 4, AR213: 4, AR289: 4, AR294: 4, AR308: 3, AR188: 3, AR285: 3, AR191: 3, AR196: 3, AR200: 3, AR189: 3, AR295: 3, AR214: 3, AR203: 3, AR291: 3, AR232: 3, AR287: 3, AR216: 3, AR272: 3, AR316: 3, AR168: 3, AR260: 3, AR210: 3, AR223: 3, AR061: 3, AR288: 2, AR060: 2, AR290: 2, AR283: 2, AR256: 2, AR055: 2, AR104: 2, AR190: 2, AR217: 2, AR211: 2, AR172: 2, AR171: 2, AR311: 2, AR253: 2, AR221: 1 S0028: 1 12 HSSFZ70 526499 22 212-280 78 Thr-17 to Leu-22. AR207: 24, AR263: 17, AR235: 15, AR195: 15, AR309: 14, AR197: 14, AR192: 13, AR201: 13, AR161: 13, AR162: 13, AR308: 12, AR213: 12, AR198: 12, AR165: 12, AR164: 12, AR311: 12, AR166: 12, AR163: 12, AR261: 11, AR193: 11, AR060: 11, AR212: 11, AR053: 11, AR089: 11, AR246: 11, AR177: 11, AR245: 11, AR039: 11, AR264: 10, AR252: 10, AR205: 10, AR170: 10, AR176: 9, AR239: 9, AR271: 9, AR282: 9, AR181: 9, AR253: 9, AR242: 9, AR223: 9, AR204: 9, AR061: 9, AR277: 9, AR312: 9, AR240: 9, AR055: 8, AR174: 8, AR229: 8, AR104: 8, AR033: 8, AR299: 8, AR214: 8, AR283: 8, AR233: 8, AR178: 8, AR316: 8, AR226: 7, AR236: 7, AR222: 7, AR227: 7, AR168: 7, AR217: 7, AR272: 7, AR169: 7, AR243: 7, AR228: 7, AR274: 7, AR250: 7, AR232: 7, AR231: 7, AR275: 6, AR182: 6, AR266: 6, AR185: 6, AR300: 6, AR171: 6, AR180: 6, AR238: 6, AR216: 6, AR179: 6, AR096: 6, AR262: 6, AR247: 6, AR183: 5, AR269: 5, AR313: 5, AR257: 5, AR196: 5, AR175: 5, AR270: 5, AR295: 5, AR254: 5, AR224: 5, AR234: 5, AR237: 5, AR230: 5, AR288: 5, AR255: 5, AR267: 5, AR225: 5, AR291: 5, AR268: 4, AR297: 4, AR172: 4, AR293: 4, AR258: 4, AR189: 4, AR188: 4, AR289: 4, AR191: 4, AR286: 4, AR199: 4, AR173: 4, AR287: 3, AR285: 3, AR296: 3, AR203: 3, AR200: 3, AR190: 3, AR294: 3, AR218: 3, AR211: 3, AR210: 2, AR290: 2, AR256: 2, AR221: 2, AR219: 2, AR260: 2, AR215: 2 H0135: 1 13 HTEIP36 520468 23  22-198 79 Glu-33 to Arg-45. AR162: 8, AR161: 7, AR163: 7, AR235: 7, AR229: 6, AR183: 6, AR176: 6, AR173: 6, AR313: 5, AR178: 5, AR266: 5, AR309: 5, AR233: 5, AR165: 5, AR181: 5, AR257: 5, AR164: 5, AR182: 5, AR274: 5, AR166: 4, AR221: 4, AR275: 4, AR175: 4, AR264: 4, AR300: 4, AR228: 4, AR268: 4, AR261: 4, AR096: 4, AR269: 4, AR293: 4, AR262: 4, AR270: 4, AR267: 4, AR196: 4, AR089: 4, AR231: 4, AR291: 4, AR238: 4, AR177: 4, AR226: 4, AR247: 4, AR282: 3, AR255: 3, AR185: 3, AR239: 3, AR299: 3, AR234: 3, AR237: 3, AR179: 3, AR277: 3, AR174: 3, AR289: 3, AR188: 3, AR258: 3, AR236: 3, AR316: 3, AR199: 3, AR225: 3, AR060: 3, AR290: 3, AR250: 3, AR227: 3, AR203: 3, AR297: 3, AR191: 3, AR294: 3, AR285: 3, AR061: 3, AR218: 3, AR296: 3, AR230: 3, AR215: 3, AR217: 3, AR172: 3, AR055: 2, AR240: 2, AR286: 2, AR288: 2, AR287: 2, AR295: 2, AR189: 2, AR224: 2, AR308: 2, AR223: 2, AR200: 2, AR263: 2, AR232: 2, AR214: 2, AR033: 2, AR190: 2, AR260: 2, AR272: 2, AR312: 2, AR104: 2, AR222: 2, AR039: 2, AR171: 2, AR219: 2, AR212: 2, AR256: 2, AR311: 1, AR243: 1, AR213: 1, AR201: 1, AR195: 1, AR169: 1, AR211: 1, AR216: 1 H0038: 1 and H0616: 1. 14 HYBAY77 520394 24 157-300 80 AR193: 6, AR204: 6, AR245: 5, AR243: 4, AR176: 4, AR162: 4, AR170: 4, AR165: 4, AR269: 4, AR250: 3, AR201: 3, AR161: 3, AR163: 3, AR246: 3, AR291: 3, AR164: 3, AR166: 3, AR197: 3, AR178: 3, AR221: 3, AR181: 3, AR282: 3, AR215: 3, AR271: 2, AR257: 2, AR266: 2, AR175: 2, AR168: 2, AR299: 2, AR264: 2, AR270: 2, AR262: 2, AR172: 2, AR055: 2, AR089: 2, AR226: 2, AR309: 2, AR312: 2, AR255: 2, AR293: 2, AR237: 2, AR277: 2, AR182: 2, AR225: 2, AR286: 2, AR217: 2, AR300: 2, AR228: 2, AR272: 2, AR231: 2, AR240: 2, AR258: 2, AR203: 2, AR233: 1, AR261: 1, AR290: 1, AR183: 1, AR268: 1, AR227: 1, AR239: 1, AR060: 1, AR232: 1, AR311: 1, AR296: 1, AR189: 1, AR216: 1, AR247: 1, AR191: 1, AR061: 1, AR294: 1, AR180: 1, AR177: 1, AR295: 1, AR188: 1, AR313: 1, AR179: 1, AR235: 1, AR104: 1, AR096: 1 H0318: 1 and H0041: 1. 15 HROAE78 520365 25 132-290 81 Ser-14 to Gln-23, AR161: 5, AR162: 5, Pro-32 to Lys-39. AR163: 5, AR176: 5, AR309: 4, AR235: 4, AR275: 4, AR182: 4, AR266: 3, AR270: 3, AR239: 3, AR257: 3, AR168: 3, AR269: 3, AR267: 3, AR228: 3, AR169: 3, AR233: 3, AR181: 3, AR178: 3, AR236: 3, AR229: 3, AR255: 3, AR238: 3, AR216: 3, AR264: 3, AR179: 3, AR055: 3, AR242: 3, AR268: 3, AR231: 3, AR175: 3, AR180: 3, AR173: 3, AR286: 3, AR237: 3, AR164: 3, AR287: 2, AR177: 2, AR230: 2, AR225: 2, AR191: 2, AR247: 2, AR226: 2, AR224: 2, AR171: 2, AR295: 2, AR291: 2, AR166: 2, AR311: 2, AR222: 2, AR294: 2, AR288: 2, AR183: 2, AR282: 2, AR261: 2, AR300: 2, AR214: 2, AR290: 2, AR196: 2, AR227: 2, AR293: 2, AR221: 2, AR277: 2, AR061: 2, AR274: 2, AR297: 2, AR246: 2, AR188: 2, AR240: 2, AR223: 2, AR165: 2, AR285: 2, AR190: 2, AR289: 2, AR313: 2, AR312: 2, AR234: 2, AR200: 2, AR272: 2, AR096: 2, AR195: 2, AR203: 1, AR189: 1, AR089: 1, AR232: 1, AR258: 1, AR256: 1, AR262: 1, AR213: 1, AR217: 1, AR212: 1, AR210: 1, AR199: 1, AR174: 1, AR260: 1, AR316: 1, AR252: 1, AR193: 1, AR060: 1, AR219: 1 H0316: 1 16 HSAVP17 519846 26  69-203 82 AR313: 28, AR089: 20, AR299: 16, AR316: 14, AR096: 14, AR185: 12, AR060: 12, AR104: 11, AR277: 8, AR165: 8, AR242: 8, AR162: 7, AR161: 7, AR163: 7, AR240: 7, AR166: 7, AR192: 7, AR245: 6, AR164: 6, AR173: 6, AR053: 5, AR264: 5, AR282: 5, AR269: 4, AR263: 4, AR274: 4, AR257: 4, AR300: 4, AR275: 4, AR193: 4, AR271: 3, AR312: 3, AR212: 3, AR283: 3, AR308: 3, AR247: 3, AR199: 3, AR205: 3, AR229: 3, AR261: 3, AR262: 3, AR235: 3, AR198: 3, AR196: 3, AR213: 2, AR178: 2, AR221: 2, AR207: 2, AR255: 2, AR311: 2, AR191: 2, AR243: 2, AR258: 2, AR176: 2, AR203: 2, AR168: 2, AR309: 2, AR183: 2, AR200: 2, AR204: 2, AR216: 2, AR189: 2, AR201: 2, AR172: 2, AR055: 2, AR253: 2, AR233: 1, AR246: 1, AR239: 1, AR195: 1, AR171: 1, AR238: 1, AR228: 1, AR222: 1, AR190: 1, AR289: 1, AR175: 1, AR188: 1, AR270: 1, AR297: 1, AR237: 1 S0114: 1 and H0402: 1. 17 HSIEA14 520387 27 141-323 83 AR169: 5, AR039: 4, AR170: 3, AR172: 3, AR235: 3, AR183: 3, AR274: 3, AR246: 2, AR192: 2, AR282: 2, AR263: 2, AR288: 2, AR243: 2, AR171: 2, AR205: 2, AR175: 2, AR222: 2, AR217: 1, AR270: 1, AR283: 1, AR096: 1, AR053: 1, AR216: 1, AR201: 1, AR225: 1, AR289: 1, AR162: 1 H0036: 1 18 HSNAQ47 847453 28  80-838 84 Lys-84 to Lys-90, AR250: 5, AR060: 5, Phe-151 to Leu- AR274: 4, AR282: 3, 156, AR225: 3, AR254: 3, Ala-204 to Asp- AR104: 3, AR089: 3, 212, AR283: 3, AR299: 3, Ala-238 to Ala-245. AR096: 3, AR240: 2, AR316: 2, AR245: 2, AR224: 2, AR180: 2, AR313: 2, AR221: 2, AR185: 2, AR277: 2, AR275: 1, AR176: 1, AR257: 1, AR205: 1 L0771: 3, L0755: 2, S0358: 1, H0587: 1, L0471: 1, H0163: 1, L0529: 1, S0152: 1, H0521: 1 and S0436: 1. 18 HSNAQ47 526779 64  85-159 120 19 HODDN65 520348 29 251-313 85 AR313: 26, AR268: 22, AR196: 21, AR173: 19, AR299: 17, AR229: 17, AR240: 16, AR300: 16, AR096: 16, AR161: 15, AR162: 15, AR180: 15, AR175: 15, AR163: 15, AR178: 15, AR247: 14, AR258: 14, AR183: 14, AR168: 14, AR267: 13, AR181: 13, AR270: 13, AR262: 13, AR257: 13, AR290: 12, AR171: 12, AR234: 12, AR174: 12, AR089: 12, AR199: 12, AR169: 12, AR238: 11, AR269: 11, AR218: 11, AR200: 11, AR179: 11, AR293: 11, AR264: 11, AR177: 11, AR223: 11, AR236: 11, AR165: 11, AR185: 11, AR188: 10, AR228: 10, AR164: 10, AR182: 10, AR203: 10, AR191: 10, AR275: 10, AR226: 10, AR166: 10, AR316: 10, AR233: 10, AR225: 10, AR189: 10, AR214: 9, AR237: 9, AR235: 9, AR213: 9, AR296: 9, AR176: 9, AR274: 9, AR261: 9, AR170: 9, AR231: 9, AR282: 9, AR255: 9, AR309: 8, AR277: 8, AR033: 8, AR053: 8, AR239: 8, AR060: 8, AR230: 8, AR294: 8, AR219: 8, AR253: 8, AR285: 8, AR260: 8, AR312: 8, AR297: 8, AR212: 7, AR263: 7, AR266: 7, AR222: 7, AR252: 7, AR291: 7, AR287: 7, AR288: 6, AR190: 6, AR039: 6, AR172: 6, AR254: 6, AR217: 6, AR286: 6, AR195: 6, AR256: 6, AR227: 6, AR308: 6, AR216: 5, AR215: 5, AR210: 5, AR295: 5, AR193: 5, AR242: 5, AR289: 5, AR245: 5, AR207: 5, AR198: 5, AR192: 5, AR104: 5, AR201: 5, AR272: 5, AR055: 5, AR271: 5, AR224: 4, AR232: 4, AR243: 4, AR311: 4, AR211: 4, AR061: 4, AR250: 4, AR283: 4, AR221: 4, AR246: 4, AR197: 4, AR205: 3, AR204: 3 H0328: 1 20 HPEAD79 520202 30  51-176 86 Lys-16 to Ser-21, AR277: 24, AR176: 5, Gly-36 to Asp-41. AR039: 5, AR162: 5, AR205: 5, AR161: 5, AR235: 5, AR163: 5, AR282: 5, AR309: 5, AR168: 4, AR223: 4, AR228: 4, AR181: 4, AR266: 4, AR182: 4, AR269: 4, AR229: 4, AR257: 3, AR233: 3, AR272: 3, AR178: 3, AR180: 3, AR165: 3, AR264: 3, AR261: 3, AR268: 3, AR195: 3, AR164: 3, AR275: 3, AR166: 3, AR267: 3, AR183: 3, AR196: 3, AR238: 3, AR237: 3, AR236: 3, AR316: 3, AR171: 3, AR170: 3, AR179: 3, AR245: 3, AR060: 3, AR262: 3, AR193: 3, AR177: 3, AR255: 3, AR242: 3, AR289: 3, AR201: 3, AR230: 3, AR311: 3, AR254: 3, AR215: 2, AR290: 2, AR294: 2, AR204: 2, AR216: 2, AR231: 2, AR287: 2, AR299: 2, AR288: 2, AR227: 2, AR300: 2, AR033: 2, AR089: 2, AR191: 2, AR239: 2, AR173: 2, AR271: 2, AR225: 2, AR270: 2, AR295: 2, AR293: 2, AR296: 2, AR234: 2, AR185: 2, AR285: 2, AR214: 2, AR226: 2, AR274: 2, AR190: 2, AR203: 2, AR096: 2, AR199: 2, AR189: 2, AR247: 2, AR297: 2, AR200: 2, AR217: 2, AR246: 2, AR055: 2, AR175: 2, AR211: 2, AR232: 2, AR061: 2, AR283: 2, AR286: 2, AR053: 1, AR222: 1, AR263: 1, AR256: 1, AR260: 1, AR258: 1, AR210: 1, AR291: 1, AR188: 1, AR174: 1, AR312: 1, AR252: 1, AR224: 1, AR219: 1 H0165: 1 21 HRDED19 526019 31  75-218 87 AR162: 3, AR163: 3, AR161: 3, AR096: 3, AR176: 3, AR204: 2, AR168: 2, AR165: 2, AR180: 2, AR282: 2, AR166: 2, AR217: 2, AR263: 2, AR221: 2, AR275: 2, AR236: 2, AR171: 2, AR214: 2, AR172: 2, AR264: 2, AR225: 1, AR288: 1, AR164: 1, AR272: 1, AR283: 1, AR274: 1, AR267: 1, AR246: 1, AR181: 1, AR199: 1, AR196: 1, AR233: 1, AR205: 1, AR285: 1, AR195: 1, AR257: 1, AR247: 1, AR089: 1 H0124: 1, L0809: 1 and H0134: 1. 22 HSAYS89 526621 32  94-225 88 AR060: 5, AR161: 4, AR162: 4, AR163: 4, AR242: 4, AR221: 4, AR176: 3, AR235: 3, AR215: 3, AR309: 3, AR178: 3, AR283: 3, AR257: 3, AR104: 3, AR264: 3, AR165: 2, AR164: 2, AR270: 2, AR089: 2, AR166: 2, AR233: 2, AR236: 2, AR228: 2, AR289: 2, AR267: 2, AR277: 2, AR293: 2, AR230: 2, AR225: 2, AR238: 2, AR175: 2, AR181: 2, AR268: 2, AR262: 2, AR286: 2, AR196: 2, AR191: 2, AR269: 2, AR229: 2, AR294: 2, AR285: 2, AR237: 2, AR240: 2, AR316: 2, AR226: 2, AR290: 2, AR171: 2, AR179: 2, AR296: 2, AR188: 2, AR291: 2, AR172: 2, AR287: 2, AR227: 2, AR182: 2, AR239: 2, AR200: 1, AR185: 1, AR174: 1, AR231: 1, AR177: 1, AR039: 1, AR261: 1, AR234: 1, AR193: 1, AR183: 1, AR288: 1, AR190: 1, AR096: 1, AR195: 1, AR061: 1, AR300: 1, AR255: 1, AR258: 1, AR201: 1, AR189: 1, AR216: 1, AR204: 1, AR299: 1, AR211: 1 S0114: 1 23 HTODK73 526021 33  43-222 89 Gln-27 to Arg-36. AR180: 12, AR181: 9, 20 AR179: 9, AR240: 8, AR229: 8, AR178: 7, AR297: 7, AR285: 7, AR235: 7, AR253: 6, AR291: 6, AR261: 6, AR204: 6, AR222: 5, AR221: 5, AR266: 5, AR288: 5, AR264: 5, AR163: 5, AR168: 5, AR309: 5, AR162: 5, AR161: 5, AR295: 5, AR214: 5, AR224: 5, AR212: 5, AR201: 5, AR300: 5, AR287: 5, AR293: 5, AR217: 5, AR223: 5, AR269: 5, AR255: 5, AR236: 5, AR311: 5, AR237: 5, AR296: 5, AR262: 5, AR183: 5, AR267: 5, AR182: 5, AR312: 5, AR290: 5, AR039: 4, AR216: 4, AR053: 4, AR215: 4, AR252: 4, AR225: 4, AR207: 4, AR172: 4, AR277: 4, AR171: 4, AR268: 4, AR165: 4, AR231: 4, AR254: 4, AR169: 4, AR230: 4, AR270: 4, AR170: 4, AR166: 4, AR286: 4, AR282: 4, AR173: 4, AR164: 4, AR289: 4, AR257: 4, AR200: 4, AR198: 4, AR175: 4, AR228: 4, AR271: 4, AR190: 4, AR177: 4, AR247: 4, AR263: 4, AR191: 4, AR205: 4, AR213: 4, AR260: 4, AR250: 4, AR233: 3, AR055: 3, AR308: 3, AR176: 3, AR188: 3, AR238: 3, AR243: 3, AR174: 3, AR234: 3, AR274: 3, AR294: 3, AR239: 3, AR226: 3, AR196: 3, AR061: 3, AR189: 3, AR299: 3, AR246: 3, AR199: 3, AR195: 3, AR219: 3, AR193: 3, AR313: 3, AR197: 3, AR211: 3, AR203: 3, AR275: 2, AR256: 2, AR089: 2, AR033: 2, AR232: 2, AR227: 2, AR316: 2, AR060: 2, AR210: 2, AR096: 2, AR258: 2, AR185: 2, AR272: 2, AR283: 2, AR218: 2, AR104: 1, AR192: 1 L0745: 7, L0771: 5, L0777: 3, L0803: 2, S0360: 1, H0351: 1, H0441: 1, H0052: 1, H0288: 1, H0424: 1, S0364: 1, H0634: 1, H0264: 1, H0494: 1, H0646: 1, L0372: 1, L0800: 1, L0764: 1, L0766: 1, L0774: 1, L0775: 1, L0805: 1, L5623: 1, L0788: 1, H0672: 1, L0746: 1, L0779: 1, L0757: 1 and S0436: 1. 24 HSVAM10 520328 34  46-201 90 AR313: 45, AR242: 40, AR192: 36, AR173: 31, AR196: 28, AR204: 26, AR258: 26, AR300: 25, AR039: 25, AR240: 25, AR247: 24, AR096: 24, AR175: 23, AR089: 23, AR229: 22, AR218: 22, AR262: 22, AR165: 21, AR185: 21, AR166: 21, AR174: 20, AR257: 20, AR179: 20, AR162: 20, AR164: 20, AR163: 20, AR161: 19, AR234: 19, AR178: 19, AR199: 19, AR293: 18, AR269: 18, AR236: 18, AR299: 17, AR183: 17, AR233: 17, AR180: 17, AR198: 17, AR270: 17, AR193: 17, AR191: 16, AR177: 16, AR181: 15, AR275: 15, AR226: 15, AR182: 15, AR268: 14, AR189: 14, AR243: 14, AR203: 14, AR238: 13, AR316: 13, AR176: 13, AR264: 13, AR296: 13, AR231: 12, AR200: 12, AR312: 12, AR287: 12, AR285: 12, AR255: 11, AR294: 11, AR237: 11, AR060: 11, AR260: 11, AR261: 11, AR219: 11, AR188: 11, AR297: 11, AR230: 11, AR205: 10, AR274: 10, AR235: 10, AR286: 10, AR197: 10, AR290: 9, AR207: 9, AR267: 9, AR266: 9, AR291: 9, AR201: 9, AR288: 9, AR295: 9, AR271: 9, AR282: 9, AR239: 9, AR195: 8, AR228: 8, AR033: 8, AR104: 8, AR277: 7, AR190: 7, AR212: 7, AR227: 7, AR263: 7, AR309: 7, AR245: 7, AR308: 6, AR213: 6, AR246: 6, AR053: 6, AR256: 5, AR289: 5, AR272: 5, AR232: 5, AR055: 4, AR061: 4, AR210: 4, AR211: 3, AR311: 3, AR283: 3, AR252: 2, AR253: 2, AR254: 2, AR250: 1, AR222: 1, AR171: 1 25 HSNBN57 526721 35 198-293 91 AR246: 3, AR217: 3, AR221: 3, AR192: 2, AR243: 2, AR171: 2, AR230: 2, AR205: 2, AR299: 2, AR161: 2, AR224: 2, AR309: 2, AR297: 2, AR089: 2, AR163: 2, AR213: 1, AR264: 1, AR257: 1, AR168: 1, AR181: 1, AR216: 1, AR222: 1, AR282: 1, AR170: 1, AR227: 1 H0163: 1 26 HSVBD22 520557 36  61-168 92 Pro-29 to Ser-35. AR311: 48, AR225: 11, AR309: 7, AR176: 7, AR282: 6, AR197: 6, AR161: 6, AR162: 6, AR170: 6, AR266: 6, AR163: 5, AR228: 5, AR182: 5, AR178: 5, AR229: 4, AR267: 4, AR172: 4, AR261: 4, AR204: 4, AR181: 4, AR275: 4, AR177: 4, AR183: 4, AR165: 4, AR268: 4, AR269: 4, AR207: 4, AR233: 4, AR289: 4, AR271: 4, AR257: 4, AR164: 4, AR291: 4, AR193: 4, AR089: 4, AR238: 4, AR166: 4, AR230: 4, AR201: 3, AR237: 3, AR245: 3, AR061: 3, AR055: 3, AR175: 3, AR293: 3, AR198: 3, AR255: 3, AR239: 3, AR226: 3, AR060: 3, AR316: 3, AR231: 3, AR168: 3, AR221: 3, AR272: 3, AR294: 3, AR234: 3, AR262: 3, AR236: 3, AR179: 3, AR263: 3, AR274: 3, AR300: 3, AR270: 3, AR227: 3, AR299: 3, AR312: 3, AR288: 3, AR223: 3, AR171: 3, AR185: 3, AR174: 2, AR247: 2, AR104: 2, AR173: 2, AR039: 2, AR287: 2, AR285: 2, AR286: 2, AR295: 2, AR290: 2, AR224: 2, AR195: 2, AR296: 2, AR232: 2, AR203: 2, AR200: 2, AR033: 2, AR211: 2, AR096: 2, AR283: 2, AR191: 2, AR190: 2, AR189: 2, AR258: 2, AR277: 2, AR216: 2, AR214: 2, AR256: 2, AR196: 1, AR205: 1, AR199: 1, AR243: 1, AR222: 1, AR246: 1, AR264: 1, AR308: 1, AR188: 1, AR240: 1, AR313: 1, AR297: 1 H0309: 1 27 HSAWA27 520302 37 319-465 93 Gly-31 to Phe-36. AR313: 65, AR089: 58, AR299: 47, AR316: 38, AR104: 34, AR096: 32, AR185: 29, AR060: 27, AR277: 20, AR240: 19, AR196: 15, AR173: 13, AR311: 12, AR264: 12, AR309: 12, AR161: 11, AR162: 11, AR163: 11, AR207: 11, AR263: 10, AR282: 10, AR213: 10, AR053: 9, AR257: 9, AR199: 9, AR242: 9, AR233: 9, AR283: 9, AR261: 9, AR191: 8, AR300: 8, AR192: 8, AR165: 8, AR262: 8, AR195: 8, AR252: 8, AR308: 8, AR164: 8, AR275: 8, AR166: 8, AR189: 7, AR212: 7, AR312: 7, AR197: 7, AR203: 7, AR245: 7, AR247: 7, AR254: 7, AR198: 7, AR255: 6, AR200: 6, AR215: 6, AR258: 6, AR170: 6, AR188: 5, AR274: 5, AR238: 5, AR296: 5, AR193: 5, AR033: 5, AR221: 5, AR293: 5, AR179: 5, AR250: 5, AR246: 5, AR169: 5, AR229: 5, AR224: 4, AR271: 4, AR253: 4, AR175: 4, AR190: 4, AR177: 4, AR205: 4, AR216: 4, AR239: 4, AR234: 4, AR204: 3, AR236: 3, AR267: 3, AR287: 3, AR226: 3, AR272: 3, AR228: 3, AR201: 3, AR289: 3, AR268: 3, AR227: 3, AR297: 3, AR231: 3, AR217: 3, AR218: 3, AR176: 3, AR260: 3, AR055: 3, AR219: 2, AR237: 2, AR270: 2, AR210: 2, AR291: 2, AR256: 2, AR269: 2, AR172: 2, AR243: 2, AR214: 2, AR288: 2, AR168: 2, AR181: 2, AR183: 2, AR285: 2, AR222: 2, AR295: 2, AR171: 2, AR061: 1, AR225: 1, AR230: 1, AR294: 1, AR290: 1, AR039: 1 S0114: 1 28 HSFAH43 520399 38 191-265 94 Asn-12 to Thr-18. AR196: 32, AR313: 22, AR165: 18, AR162: 18, AR161: 18, AR164: 18, AR089: 18, AR163: 17, AR166: 17, AR181: 17, AR174: 16, AR175: 16, AR173: 14, AR178: 14, AR299: 14, AR182: 14, AR309: 13, AR053: 12, AR293: 12, AR177: 12, AR191: 12, AR257: 11, AR226: 11, AR316: 11, AR300: 11, AR189: 11, AR236: 11, AR247: 11, AR261: 10, AR176: 10, AR312: 10, AR268: 10, AR258: 10, AR185: 10, AR179: 10, AR096: 9, AR060: 9, AR212: 9, AR199: 9, AR264: 9, AR180: 9, AR270: 9, AR183: 9, AR262: 9, AR240: 9, AR203: 8, AR296: 8, AR229: 8, AR200: 8, AR269: 8, AR266: 8, AR238: 8, AR308: 8, AR188: 8, AR235: 8, AR267: 8, AR239: 8, AR233: 7, AR275: 7, AR263: 7, AR197: 7, AR255: 7, AR274: 7, AR277: 7, AR285: 7, AR228: 7, AR295: 7, AR286: 7, AR104: 7, AR294: 7, AR297: 6, AR198: 6, AR254: 6, AR213: 6, AR218: 6, AR290: 6, AR190: 6, AR271: 6, AR288: 5, AR272: 5, AR230: 5, AR204: 5, AR287: 5, AR231: 5, AR289: 5, AR234: 5, AR291: 5, AR215: 5, AR237: 5, AR195: 5, AR260: 5, AR282: 5, AR192: 4, AR061: 4, AR172: 4, AR193: 4, AR232: 4, AR227: 4, AR033: 4, AR253: 4, AR219: 4, AR256: 4, AR205: 4, AR211: 4, AR201: 3, AR055: 3, AR242: 3, AR210: 3, AR225: 3, AR039: 3, AR283: 3, AR250: 3, AR311: 2, AR216: 2, AR246: 2 H0154: 1 29 HSPAA60 526447 39 198-338 95 AR215: 5, AR204: 4, AR282: 4, AR224: 3, AR163: 3, AR162: 3, AR161: 3, AR168: 2, AR309: 2, AR270: 2, AR263: 2, AR311: 2, AR271: 2, AR269: 2, AR182: 2, AR225: 2, AR089: 2, AR176: 2, AR246: 2, AR201: 1, AR183: 1, AR181: 1, AR222: 1, AR212: 1, AR264: 1, AR165: 1, AR214: 1, AR164: 1, AR238: 1, AR178: 1, AR096: 1, AR171: 1, AR267: 1, AR228: 1, AR266: 1, AR294: 1, AR242: 1, AR199: 1, AR216: 1 L0805: 1 and H0478: 1. 30 HFAEF57 534142 40 232-492 96 Leu-69 to Leu-74. AR241: 14, AR161: 14, AR162: 13, AR163: 13, AR313: 10, AR242: 10, AR201: 10, AR165: 9, AR164: 9, AR252: 9, AR197: 9, AR194: 9, AR053: 9, AR166: 9, AR198: 8, AR245: 8, AR236: 8, AR192: 8, AR176: 8, AR206: 8, AR250: 8, AR212: 8, AR235: 8, AR196: 7, AR039: 7, AR271: 7, AR186: 7, AR052: 7, AR173: 7, AR204: 7, AR246: 7, AR253: 7, AR263: 7, AR207: 7, AR191: 7, AR275: 7, AR180: 7, AR226: 7, AR272: 7, AR247: 7, AR089: 6, AR181: 6, AR299: 6, AR195: 6, AR293: 6, AR244: 6, AR193: 6, AR312: 6, AR213: 6, AR229: 6, AR251: 6, AR188: 6, AR055: 6, AR202: 6, AR309: 6, AR287: 6, AR264: 6, AR238: 6, AR273: 6, AR300: 6, AR174: 6, AR177: 6, AR240: 6, AR257: 6, AR237: 5, AR243: 5, AR061: 5, AR233: 5, AR228: 5, AR261: 5, AR184: 5, AR182: 5, AR262: 5, AR185: 5, AR096: 5, AR270: 5, AR189: 5, AR190: 5, AR274: 5, AR248: 5, AR205: 5, AR183: 5, AR175: 5, AR288: 5, AR169: 5, AR033: 5, AR297: 5, AR269: 5, AR199: 5, AR178: 5, AR249: 5, AR295: 5, AR308: 4, AR223: 4, AR254: 4, AR060: 4, AR104: 4, AR216: 4, AR296: 4, AR282: 4, AR227: 4, AR290: 4, AR221: 4, AR266: 4, AR232: 4, AR239: 4, AR311: 4, AR179: 4, AR298: 4, AR200: 4, AR231: 4, AR285: 4, AR255: 4, AR268: 4, AR286: 4, AR267: 4, AR230: 4, AR316: 4, AR294: 4, AR214: 4, AR277: 4, AR234: 4, AR168: 4, AR258: 3, AR291: 3, AR170: 3, AR217: 3, AR203: 3, AR292: 3, AR171: 3, AR289: 3, AR310: 3, AR218: 3, AR265: 3, AR215: 3, AR259: 3, AR284: 3, AR283: 2, AR225: 2, AR222: 2, AR219: 2, AR260: 2, AR210: 2, AR172: 2, AR224: 2, AR211: 1, AR256: 1 S6024: 1 31 HEGAH43 532596 41  29-364 97 Lys-35 to Glu-41, AR161: 7, AR163: 6, 20p13 192340, Ala-62 to Asn-67. AR162: 6, AR176: 6, 234200 AR263: 4, AR275: 4, AR269: 4, AR266: 4, AR216: 4, AR214: 4, AR183: 4, AR192: 4, AR233: 4, AR235: 4, AR270: 4, AR267: 4, AR228: 4, AR309: 4, AR261: 3, AR172: 3, AR236: 3, AR272: 3, AR264: 3, AR182: 3, AR217: 3, AR288: 3, AR293: 3, AR257: 3, AR274: 3, AR178: 3, AR169: 3, AR245: 3, AR229: 3, AR255: 3, AR311: 3, AR268: 3, AR177: 3, AR294: 3, AR262: 3, AR166: 3, AR179: 3, AR175: 3, AR170: 3, AR224: 3, AR287: 3, AR164: 3, AR282: 3, AR238: 3, AR239: 3, AR171: 3, AR191: 3, AR237: 3, AR061: 3, AR300: 3, AR291: 3, AR221: 2, AR181: 2, AR234: 2, AR173: 2, AR196: 2, AR231: 2, AR240: 2, AR252: 2, AR285: 2, AR286: 2, AR190: 2, AR290: 2, AR168: 2, AR055: 2, AR174: 2, AR185: 2, AR289: 2, AR165: 2, AR308: 2, AR227: 2, AR295: 2, AR223: 2, AR232: 2, AR188: 2, AR297: 2, AR201: 2, AR256: 2, AR189: 2, AR104: 2, AR200: 2, AR247: 2, AR226: 2, AR060: 2, AR225: 2, AR089: 1, AR230: 1, AR312: 1, AR258: 1, AR211: 1, AR199: 1, AR277: 1, AR210: 1, AR316: 1, AR212: 1, AR180: 1, AR219: 1, AR203: 1, AR033: 1, AR299: 1, AR260: 1 L0758: 5, H0550: 1, S0374: 1 and L0779: 1. 32 HAGDG59 534165 42 124-1026 98 Lys-29 to Val-34, AR039: 36, AR299:  4 Cys-94 to Asp-99, 24, AR251: 24, AR206: Ser-102 to Val-107, 23, AR205: 21, AR248: Gln-133 to Lys- 20, AR252: 20, AR244: 139. 19, AR238: 18, AR186: 18, AR254: 16, AR263: 14, AR207: 14, AR250: 14, AR275: 13, AR249: 13, AR264: 13, AR246: 12, AR181: 12, AR241: 12, AR204: 11, AR274: 11, AR269: 11, AR202: 11, AR185: 10, AR253: 10, AR243: 10, AR292: 10, AR052: 9, AR265: 9, AR310: 9, AR060: 9, AR316: 9, AR309: 9, AR191: 9, AR190: 9, AR273: 8, AR161: 8, AR268: 8, AR162: 8, AR270: 8, AR189: 8, AR163: 8, AR240: 8, AR053: 8, AR312: 8, AR089: 8, AR226: 8, AR096: 7, AR033: 7, AR290: 7, AR183: 7, AR237: 7, AR194: 7, AR177: 7, AR198: 7, AR174: 7, AR313: 7, AR201: 7, AR271: 7, AR104: 7, AR192: 7, AR175: 6, AR272: 6, AR213: 6, AR291: 6, AR239: 6, AR179: 6, AR235: 6, AR165: 6, AR061: 6, AR296: 6, AR308: 6, AR164: 5, AR267: 5, AR188: 5, AR284: 5, AR227: 5, AR166: 5, AR298: 5, AR176: 5, AR266: 5, AR178: 5, AR182: 5, AR234: 5, AR212: 5, AR295: 4, AR277: 4, AR193: 4, AR282: 4, AR300: 4, AR293: 4, AR232: 4, AR229: 4, AR285: 4, AR311: 4, AR196: 4, AR055: 4, AR231: 4, AR247: 4, AR173: 3, AR283: 3, AR184: 3, AR245: 3, AR233: 3, AR203: 3, AR197: 3, AR289: 3, AR257: 3, AR261: 3, AR294: 3, AR297: 2, AR242: 2, AR217: 2, AR288: 2, AR286: 2, AR218: 2, AR255: 2, AR195: 2, AR259: 2, AR256: 2, AR200: 2, AR219: 2, AR180: 2, AR228: 2, AR210: 2, AR199: 2, AR224: 1, AR211: 1, AR230: 1, AR236: 1, AR287: 1 S0422: 22, S0408: 9, L0659: 9, S0438: 8, S0354: 6, L0754: 6, S0126: 5, H0543: 5, S0358: 4, S0444: 4, S0406: 4, H0436: 4, L0740: 4, L0777: 4, H0144: 3, S0374: 3, L0750: 3, L0599: 3, H0170: 2, H0717: 2, H0740: 2, S0360: 2, S0410: 2, H0747: 2, H0749: 2, H0587: 2, H0574: 2, H0486: 2, H0575: 2, H0036: 2, S0003: 2, H0622: 2, L0475: 2, H0509: 2, L0667: 2, L0771: 2, L0662: 2, L0766: 2, L0804: 2, L0790: 2, H0710: 2, L0748: 2, L0745: 2, L0749: 2, L0731: 2, S0026: 2, H0422: 2, H0171: 1, H0686: 1, S0040: 1, H0716: 1, L0785: 1, L2991: 1, S0212: 1, S0442: 1, H0393: 1, L0717: 1, H0441: 1, H0497: 1, H0427: 1, H0590: 1, S0346: 1, S0474: 1, H0581: 1, H0746: 1, H0050: 1, H0239: 1, H0510: 1, H0266: 1, H0553: 1, H0169: 1, H0264: 1, H0494: 1, S0450: 1, S0440: 1, H0654: 1, H0652: 1, S0344: 1, H0529: 1, H0026: 1, L0371: 1, L0372: 1, L0764: 1, L0521: 1, L0768: 1, L0649: 1, L0652: 1, L0653: 1, L0809: 1, L0367: 1, L0663: 1, L0665: 1, S0428: 1, H0699: 1, H0547: 1, H0670: 1, H0660: 1, S0330: 1, S0378: 1, H0518: 1, H0521: 1, H0522: 1, S0028: 1, L0744: 1, L0439: 1, L0751: 1, S0031: 1, S0260: 1, L0581: 1, L0362: 1, H0136: 1, S0276: 1, H0506: 1 and H0721: 1. 33 HNGBX63 532615 43 120-434 99 Pro-46 to Pro-53, AR170: 5, AR309: 3, His-55 to Cys-63. AR242: 3, AR282: 3, AR225: 3, AR193: 3, AR192: 3, AR214: 2, AR180: 2, AR172: 2, AR039: 2, AR311: 2, AR271: 2, AR053: 2, AR264: 2, AR300: 1, AR196: 1, AR178: 1, AR272: 1, AR277: 1, AR270: 1, AR237: 1, AR161: 1 S0052: 1 34 HE2AG50 532595 44  19-150 100 Ser-37 to Tyr-43. AR252: 4, AR250: 4, AR289: 3, AR282: 3, AR169: 3, AR296: 3, AR180: 2, AR178: 2, AR183: 2, AR246: 2, AR168: 2, AR255: 2, AR185: 2, AR264: 2, AR213: 2, AR240: 2, AR309: 2, AR257: 2, AR233: 2, AR274: 1, AR215: 1, AR222: 1, AR216: 1 H0170: 1, H0574: 1, H0052: 1, H0048: 1 and L0766: 1. 35 HCUIN80 534128 45 106-255 101 Pro-26 to Arg-40, AR313: 9, AR173: 8, Pro-43 to Lys-49. AR161: 7, AR162: 7, AR163: 7, AR165: 5, AR164: 5, AR258: 5, AR236: 5, AR166: 5, AR089: 5, AR175: 5, AR196: 5, AR262: 5, AR096: 5, AR180: 5, AR264: 5, AR257: 4, AR247: 4, AR185: 4, AR240: 4, AR300: 4, AR299: 4, AR274: 4, AR263: 4, AR312: 4, AR177: 4, AR275: 4, AR218: 4, AR053: 4, AR234: 3, AR174: 3, AR191: 3, AR282: 3, AR229: 3, AR212: 3, AR293: 3, AR261: 3, AR269: 3, AR170: 3, AR199: 3, AR182: 3, AR179: 3, AR183: 3, AR277: 3, AR203: 3, AR060: 3, AR316: 3, AR198: 3, AR181: 3, AR200: 3, AR233: 3, AR311: 3, AR226: 3, AR238: 3, AR270: 3, AR286: 3, AR189: 3, AR294: 2, AR283: 2, AR104: 2, AR178: 2, AR296: 2, AR193: 2, AR285: 2, AR291: 2, AR219: 2, AR287: 2, AR289: 2, AR309: 2, AR237: 2, AR288: 2, AR225: 2, AR033: 2, AR295: 2, AR039: 2, AR231: 2, AR255: 2, AR172: 2, AR213: 2, AR176: 2, AR268: 2, AR197: 2, AR242: 2, AR260: 2, AR205: 2, AR195: 2, AR228: 2, AR230: 2, AR188: 2, AR297: 2, AR267: 1, AR266: 1, AR204: 1, AR227: 1, AR239: 1, AR290: 1, AR201: 1, AR252: 1, AR192: 1, AR190: 1, AR256: 1 H0402: 1 36 HADCL29 532056 46 248-391 102 AR215: 6, AR269: 3, AR253: 3, AR282: 3, AR053: 3, AR235: 3, AR172: 3, AR195: 3, AR170: 3, AR309: 2, AR264: 2, AR311: 2, AR225: 2, AR270: 2, AR266: 2, AR228: 2, AR296: 2, AR183: 2, AR237: 2, AR275: 2, AR268: 2, AR216: 2, AR230: 2, AR205: 1, AR199: 1, AR261: 1, AR181: 1, AR238: 1, AR166: 1, AR210: 1, AR231: 1, AR234: 1, AR313: 1, AR229: 1, AR274: 1, AR089: 1, AR277: 1, AR247: 1, AR061: 1, AR104: 1, AR096: 1 H0427: 1, L0748: 1 and L0754: 1. 37 HAPPS89 532135 47  54-353 103 AR313: 73, AR300: 52, AR192: 52, AR161: 51, AR163: 51, AR162: 51, AR275: 51, AR264: 51, AR242: 44, AR274: 43, AR193: 42, AR240: 38, AR174: 35, AR053: 34, AR247: 33, AR185: 33, AR229: 33, AR212: 33, AR312: 31, AR177: 31, AR165: 31, AR195: 31, AR196: 30, AR233: 30, AR164: 30, AR166: 29, AR234: 29, AR270: 28, AR269: 28, AR213: 28, AR238: 27, AR254: 27, AR096: 26, AR181: 26, AR198: 25, AR263: 25, AR237: 24, AR236: 24, AR179: 23, AR089: 23, AR039: 23, AR226: 22, AR268: 21, AR207: 21, AR230: 21, AR173: 20, AR239: 20, AR191: 20, AR308: 20, AR204: 19, AR183: 18, AR250: 18, AR104: 18, AR197: 18, AR277: 17, AR180: 17, AR033: 17, AR189: 16, AR253: 16, AR252: 16, AR311: 16, AR175: 16, AR235: 16, AR228: 15, AR257: 15, AR182: 15, AR272: 15, AR178: 15, AR231: 15, AR267: 15, AR282: 15, AR201: 15, AR293: 14, AR199: 14, AR271: 14, AR188: 13, AR299: 13, AR227: 13, AR245: 13, AR176: 13, AR243: 12, AR262: 12, AR258: 12, AR203: 12, AR287: 11, AR296: 11, AR316: 11, AR286: 11, AR309: 11, AR200: 11, AR060: 10, AR190: 10, AR285: 10, AR290: 10, AR291: 10, AR266: 10, AR205: 9, AR288: 9, AR297: 9, AR232: 9, AR294: 9, AR218: 8, AR261: 7, AR289: 7, AR246: 7, AR169: 6, AR283: 6, AR295: 6, AR255: 6, AR219: 6, AR055: 5, AR171: 5, AR061: 5, AR223: 4, AR260: 4, AR168: 4, AR222: 3, AR170: 3, AR217: 3, AR216: 3, AR256: 3, AR214: 2, AR224: 2, AR225: 2, AR172: 2, AR211: 2, AR210: 2, AR215: 1 H0575: 1 38 HFGAH44 533132 48  34-210 104 Pro-30 to Gln-35, AR235: 7, AR269: 7, Pro-44 to Leu-50. AR181: 6, AR257: 5, AR161: 5, AR162: 5, AR236: 5, AR178: 5, AR165: 5, AR182: 5, AR163: 5, AR287: 5, AR164: 5, AR291: 5, AR255: 5, AR191: 5, AR166: 5, AR297: 5, AR261: 5, AR179: 5, AR173: 5, AR228: 5, AR176: 5, AR290: 5, AR172: 4, AR285: 4, AR293: 4, AR267: 4, AR183: 4, AR268: 4, AR294: 4, AR270: 4, AR309: 4, AR266: 4, AR264: 4, AR240: 4, AR215: 4, AR262: 4, AR231: 4, AR289: 4, AR295: 4, AR224: 4, AR180: 4, AR233: 4, AR174: 4, AR177: 4, AR096: 4, AR238: 4, AR175: 4, AR247: 4, AR308: 4, AR169: 4, AR272: 3, AR296: 3, AR313: 3, AR200: 3, AR196: 3, AR237: 3, AR286: 3, AR229: 3, AR258: 3, AR316: 3, AR216: 3, AR260: 3, AR300: 3, AR199: 3, AR188: 3, AR190: 3, AR275: 3, AR225: 3, AR234: 3, AR312: 3, AR168: 3, AR217: 3, AR223: 3, AR060: 3, AR221: 3, AR288: 3, AR189: 3, AR089: 3, AR299: 3, AR033: 3, AR230: 3, AR239: 3, AR210: 3, AR246: 3, AR039: 2, AR226: 2, AR243: 2, AR061: 2, AR104: 2, AR277: 2, AR170: 2, AR055: 2, AR282: 2, AR203: 2, AR227: 2, AR185: 2, AR274: 2, AR222: 2, AR171: 2, AR219: 2, AR250: 2, AR311: 2, AR254: 2, AR232: 2, AR214: 2, AR253: 2, AR213: 1, AR256: 1, AR218: 1, AR283: 1, AR211: 1, AR271: 1 L0439: 6, L0758: 5, L0766: 3, L0803: 3, L0754: 3, L0749: 3, S0010: 2, H0616: 2, L0775: 2, L0438: 2, L0777: 2, H0543: 2, H0656: 1, H0581: 1, H0178: 1, H0051: 1, H0647: 1, L0761: 1, L0521: 1, L0794: 1, L0784: 1, L0653: 1, L0809: 1, L0791: 1, L0663: 1, L0664: 1, H0690: 1, H0658: 1, H0696: 1, L0780: 1 and L0755: 1. 39 HFIHZ96 532046 49  39-233 105 Asn-30 to Gly-37. AR221: 4, AR170: 3, AR296: 3, AR183: 3, AR163: 2, AR176: 2, AR282: 2, AR240: 2, AR224: 2, AR225: 2, AR297: 2, AR270: 2, AR180: 2, AR274: 2, AR104: 2, AR222: 2, AR264: 2, AR228: 2, AR232: 2, AR213: 2, AR229: 2, AR311: 1, AR313: 1, AR312: 1, AR230: 1, AR247: 1, AR238: 1, AR089: 1, AR096: 1, AR286: 1, AR190: 1, AR252: 1, AR277: 1, AR171: 1 H0728: 1, L0643: 1 and S0194: 1. 40 HFIUR10 532060 50  50-184 106 Gln-31 to Pro-39. AR169: 4, AR165: 4, AR161: 3, AR163: 3, AR215: 3, AR162: 3, AR166: 3, AR246: 3, AR252: 3, AR313: 3, AR089: 3, AR311: 3, AR266: 2, AR270: 2, AR180: 2, AR261: 2, AR164: 2, AR224: 2, AR269: 2, AR096: 2, AR236: 2, AR289: 2, AR201: 2, AR297: 2, AR312: 2, AR205: 2, AR217: 2, AR255: 2, AR172: 2, AR240: 2, AR216: 2, AR183: 2, AR309: 2, AR173: 2, AR291: 2, AR176: 2, AR196: 2, AR295: 1, AR264: 1, AR225: 1, AR299: 1, AR033: 1, AR174: 1, AR257: 1, AR282: 1, AR060: 1, AR230: 1, AR178: 1, AR177: 1, AR316: 1, AR168: 1, AR243: 1, AR283: 1, AR268: 1, AR277: 1, AR189: 1, AR290: 1, AR247: 1, AR055: 1, AR308: 1, AR288: 1, AR300: 1, AR237: 1, AR185: 1 H0265: 2, L0591: 2, H0556: 1, S0356: 1, H0271: 1, H0622: 1, S0428: 1, S0434: 1 and S0196: 1. 41 HLDNA86 535730 51  45-323 107 Arg-35 to Ala-41. AR186: 162, AR298: 16 160, AR241: 158, AR259: 155, AR227: 154, AR233: 143, AR275: 136, AR055: 135, AR232: 134, AR192: 131, AR061: 131, AR175: 130, AR284: 127, AR194: 125, AR204: 120, AR295: 120, AR237: 117, AR285: 116, AR033: 112, AR286: 111, AR300: 109, AR184: 108, AR185: 107, AR226: 107, AR238: 107, AR293: 105, AR177: 100, AR198: 100, AR231: 100, AR299: 100, AR206: 99, AR202: 97, AR271: 95, AR052: 94, AR273: 91, AR104: 84, AR243: 84, AR179: 83, AR292: 80, AR294: 77, AR291: 71, AR282: 70, AR229: 68, AR244: 67, AR283: 67, AR267: 63, AR234: 63, AR053: 61, AR182: 60, AR310: 57, AR205: 57, AR039: 56, AR183: 56, AR274: 56, AR089: 52, AR060: 51, AR247: 49, AR296: 48, AR251: 46, AR240: 46, AR269: 46, AR309: 45, AR316: 43, AR213: 43, AR258: 42, AR246: 39, AR289: 38, AR265: 38, AR218: 38, AR290: 37, AR268: 36, AR270: 36, AR211: 35, AR219: 35, AR277: 34, AR256: 34, AR312: 33, AR161: 32, AR162: 32, AR228: 31, AR096: 29, AR163: 29, AR239: 29, AR266: 29, AR248: 28, AR262: 27, AR203: 27, AR174: 27, AR199: 26, AR249: 25, AR230: 25, AR287: 23, AR195: 23, AR311: 22, AR191: 22, AR190: 22, AR263: 21, AR236: 21, AR313: 21, AR288: 21, AR181: 20, AR253: 20, AR257: 20, AR196: 19, AR193: 19, AR188: 19, AR201: 19, AR210: 19, AR255: 17, AR176: 17, AR200: 17, AR189: 17, AR264: 17, AR178: 17, AR272: 16, AR260: 15, AR261: 14, AR173: 14, AR297: 14, AR197: 14, AR212: 12, AR180: 12, AR165: 11, AR164: 11, AR235: 11, AR166: 10, AR207: 10, AR254: 9, AR250: 9, AR221: 7, AR245: 7, AR216: 7, AR308: 6, AR242: 6, AR169: 6, AR215: 6, AR217: 5, AR252: 5, AR172: 4, AR225: 4, AR224: 4, AR223: 3, AR222: 3, AR214: 2, AR168: 2, AR171: 2, AR170: 1 L0747: 13, L0731: 10, L0758: 10, L0769: 7, L0740: 7, H0545: 6, L0803: 6, L0809: 6, L0755: 6, S0360: 5, L0775: 5, L0748: 5, L0749: 5, H0305: 4, S0358: 4, H0266: 4, S0126: 4, L0757: 4, H0556: 3, H0740: 3, H0722: 3, H0530: 3, S0438: 3, L0771: 3, L0774: 3, L0657: 3, L0782: 3, L0789: 3, S0192: 3, H0171: 2, H0265: 2, S0040: 2, T0049: 2, H0638: 2, S0418: 2, S0278: 2, H0574: 2, H0486: 2, S0280: 2, H0309: 2, H0046: 2, H0616: 2, H0652: 2, S0142: 2, L0770: 2, L0768: 2, L0794: 2, L0518: 2, L0666: 2, H0658: 2, S3014: 2, S0027: 2, L0743: 2, L0751: 2, L0756: 2, L0753: 2, L0759: 2, L0593: 2, S0194: 2, H0624: 1, H0149: 1, H0717: 1, H0295: 1, H0294: 1, S0218: 1, H0656: 1, S0298: 1, H0484: 1, H0661: 1, H0228: 1, S0356: 1, S0442: 1, S0408: 1, H0730: 1, H0747: 1, H0393: 1, H0549: 1, H0441: 1, H0586: 1, H0333: 1, T0040: 1, H0575: 1, H0318: 1, S0474: 1, H0196: 1, H0746: 1, L0738: 1, H0544: 1, H0041: 1, H0571: 1, H0123: 1, H0081: 1, H0620: 1, H0024: 1, H0510: 1, H0687: 1, S0314: 1, S0003: 1, S0022: 1, H0615: 1, H0622: 1, T0006: 1, H0644: 1, H0617: 1, H0673: 1, H0708: 1, H0135: 1, H0163: 1, H0634: 1, H0551: 1, T0067: 1, H0100: 1, H0561: 1, H0641: 1, S0144: 1, S0422: 1, H0695: 1, L0520: 1, L0763: 1, L0638: 1, L5565: 1, L0373: 1, L0800: 1, L0641: 1, L0644: 1, L0648: 1, L0766: 1, L0804: 1, L0375: 1, L0805: 1, L0653: 1, L0776: 1, L0659: 1, L0783: 1, L5622: 1, L0663: 1, L0665: 1, H0684: 1, H0651: 1, S0378: 1, H0518: 1, S0044: 1, H0555: 1, S3012: 1, S0390: 1, S0206: 1, L0741: 1, L0750: 1, H0445: 1, S0436: 1, S0026: 1, H0653: 1, H0543: 1, H0423: 1, H0422: 1, S0462: 1 and H0721: 1. 42 HNGAN75 532613 52  41-118 108 AR180: 4, AR266: 4, AR263: 4, AR225: 4, AR176: 4, AR235: 3, AR162: 3, AR161: 3, AR163: 3, AR217: 3, AR181: 3, AR272: 3, AR221: 2, AR274: 2, AR193: 2, AR224: 2, AR288: 2, AR269: 2, AR309: 2, AR053: 2, AR290: 2, AR171: 2, AR228: 2, AR257: 2, AR178: 1, AR261: 1, AR262: 1, AR195: 1, AR277: 1, AR237: 1, AR293: 1, AR190: 1, AR255: 1, AR254: 1, AR227: 1, AR264: 1, AR231: 1, AR200: 1, AR199: 1, AR286: 1, AR170: 1, AR226: 1, AR179: 1, AR175: 1, AR182: 1, AR287: 1, AR289: 1, AR233: 1, AR268: 1 S0052: 1 43 HCUIO20 534220 53  43-246 109 Ser-41 to Lys-49. AR266: 4, AR252: 4, AR263: 4, AR170: 3, AR282: 3, AR219: 3, AR225: 3, AR254: 3, AR264: 3, AR053: 2, AR294: 2, AR274: 2, AR221: 2, AR308: 2, AR233: 2, AR214: 2, AR299: 2, AR165: 2, AR180: 2, AR227: 2, AR239: 2, AR257: 2, AR164: 2, AR205: 2, AR272: 2, AR217: 2, AR039: 2, AR291: 1, AR166: 1, AR193: 1, AR261: 1, AR224: 1, AR312: 1, AR222: 1, AR199: 1, AR060: 1, AR311: 1, AR218: 1, AR089: 1, AR267: 1 H0402: 1 44 HLTEF12 532354 54 686-820 110 Cys-31 to Trp-36. AR235: 39, AR261: 15q26.1 166800, 23, AR216: 23, AR266: 210900 21, AR217: 20, AR291: 20, AR286: 19, AR215: 19, AR283: 18, AR256: 16, AR289: 15, AR258: 15, AR295: 15, AR236: 15, AR263: 14, AR214: 14, AR260: 14, AR257: 14, AR172: 13, AR264: 13, AR221: 12, AR285: 12, AR309: 12, AR170: 10, AR171: 10, AR297: 10, AR188: 10, AR288: 9, AR053: 9, AR168: 9, AR262: 9, AR287: 9, AR169: 9, AR294: 9, AR224: 8, AR255: 8, AR231: 8, AR165: 8, AR164: 8, AR055: 8, AR282: 7, AR166: 7, AR250: 7, AR201: 7, AR308: 7, AR293: 7, AR271: 7, AR222: 7, AR207: 6, AR197: 6, AR311: 6, AR272: 6, AR161: 6, AR162: 6, AR163: 6, AR245: 6, AR182: 6, AR246: 6, AR200: 6, AR312: 5, AR313: 5, AR296: 5, AR239: 5, AR232: 5, AR176: 5, AR198: 5, AR243: 5, AR199: 5, AR196: 5, AR300: 5, AR195: 5, AR212: 5, AR205: 5, AR203: 4, AR247: 4, AR225: 4, AR226: 4, AR254: 4, AR237: 4, AR233: 4, AR234: 4, AR253: 4, AR213: 4, AR268: 4, AR191: 4, AR183: 4, AR218: 4, AR228: 4, AR316: 4, AR060: 3, AR193: 3, AR061: 3, AR229: 3, AR269: 3, AR089: 3, AR267: 3, AR039: 3, AR277: 3, AR275: 3, AR177: 3, AR240: 3, AR173: 3, AR219: 3, AR230: 3, AR211: 3, AR274: 3, AR270: 3, AR189: 2, AR290: 2, AR192: 2, AR223: 2, AR238: 2, AR175: 2, AR096: 2, AR181: 2, AR174: 2, AR299: 2, AR033: 2, AR104: 2, AR178: 2, AR190: 2, AR210: 1, AR179: 1, AR185: 1, AR227: 1 H0617: 24, S0436: 12, S0410: 7, H0620: 7, H0706: 6, L0604: 6, L0774: 5, L0665: 5, H0341: 4, H0484: 4, H0574: 4, L0163: 4, S0440: 4, L5622: 4, L0666: 4, H0521: 4, L0439: 4, L0747: 4, S0358: 3, S0408: 3, S0046: 3, H0370: 3, L0622: 3, H0013: 3, S0010: 3, H0041: 3, H0009: 3, L0471: 3, H0012: 3, H0199: 3, S0051: 3, H0083: 3, H0644: 3, H0090: 3, T0041: 3, L0770: 3, L0769: 3, L0646: 3, L0519: 3, L0663: 3, L3826: 3, L0750: 3, L0777: 3, L0752: 3, L0755: 3, L0731: 3, S0434: 3, L0588: 3, H0542: 3, H0543: 3, L0615: 2, S0116: 2, H0255: 2, H0729: 2, H0735: 2, H0208: 2, H0619: 2, H0549: 2, L3816: 2, S0474: 2, H0581: 2, H0046: 2, H0024: 2, H0266: 2, H0553: 2, H0181: 2, H0606: 2, H0708: 2, S0366: 2, H0087: 2, S0038: 2, H0100: 2, H0494: 2, H0560: 2, S0144: 2, S0426: 2, L0763: 2, L0372: 2, L0764: 2, L0662: 2, L0655: 2, L0783: 2, H0144: 2, H0702: 2, H0520: 2, S0126: 2, S0380: 2, S0406: 2, L0742: 2, L0779: 2, L0759: 2, H0445: 2, L0596: 2, L0592: 2, L0599: 2, L0608: 2, L0601: 2, H0423: 2, H0422: 2, H0265: 1, S0040: 1, H0713: 1, H0717: 1, H0716: 1, H0740: 1, H0295: 1, H0294: 1, H0650: 1, H0657: 1, H0656: 1, S0180: 1, S0029: 1, H0483: 1, H0664: 1, H0306: 1, H0305: 1, S0418: 1, S0420: 1, S0442: 1, S0376: 1, S0444: 1, S0360: 1, H0675: 1, H0676: 1, H0637: 1, H0580: 1, H0742: 1, H0728: 1, H0733: 1, S0045: 1, S0476: 1, H0645: 1, H0431: 1, H0643: 1, H0632: 1, L0623: 1, H0486: 1, T0109: 1, S0280: 1, H0156: 1, H0599: 1, H0575: 1, H0618: 1, H0318: 1, S0049: 1, H0746: 1, H0052: 1, H0597: 1, H0545: 1, H0562: 1, H0172: 1, H0050: 1, H0154: 1, S0362: 1, H0018: 1, H0095: 1, H0373: 1, N0004: 1, T0010: 1, H0355: 1, H0099: 1, S6028: 1, S0312: 1, S0314: 1, L0483: 1, H0604: 1, H0213: 1, H0031: 1, H0111: 1, H0032: 1, S0364: 1, H0124: 1, H0551: 1, H0272: 1, S0112: 1, L0354: 1, L0370: 1, H0745: 1, S0438: 1, H0509: 1, S0344: 1, S0422: 1, L0598: 1, L0640: 1, L5566: 1, L0761: 1, L0374: 1, L0771: 1, L0773: 1, L0768: 1, L0775: 1, L0776: 1, L0629: 1, L0657: 1, L0384: 1, L0809: 1, L0530: 1, L0710: 1, H0698: 1, H0701: 1, H0703: 1, H0725: 1, H0723: 1, H0724: 1, L3811: 1, L3825: 1, H0547: 1, H0593: 1, H0435: 1, H0659: 1, H0658: 1, H0670: 1, H0660: 1, H0648: 1, L0355: 1, H0436: 1, H0626: 1, L0748: 1, L0593: 1, L0361: 1, S0011: 1, H0136: 1, H0216: 1, H0506: 1 and H0721: 1. 45 HCFBJ91 531960 55  61-219 111 AR313: 19, AR096: 13, AR165: 12, AR247: 12, AR164: 12, AR163: 11, AR166: 11, AR161: 11, AR162: 11, AR173: 10, AR089: 10, AR240: 10, AR183: 9, AR300: 9, AR180: 9, AR229: 9, AR299: 8, AR179: 8, AR293: 8, AR274: 8, AR178: 7, AR264: 7, AR296: 7, AR226: 7, AR175: 7, AR242: 7, AR262: 7, AR257: 7, AR185: 7, AR270: 7, AR234: 7, AR181: 7, AR312: 7, AR252: 7, AR174: 6, AR272: 6, AR196: 6, AR238: 6, AR263: 6, AR269: 6, AR258: 6, AR285: 6, AR261: 6, AR297: 6, AR275: 6, AR316: 6, AR199: 6, AR200: 6, AR268: 6, AR233: 5, AR214: 5, AR216: 5, AR039: 5, AR235: 5, AR282: 5, AR309: 5, AR182: 5, AR231: 5, AR060: 5, AR104: 5, AR237: 5, AR176: 5, AR215: 5, AR053: 5, AR287: 5, AR189: 4, AR171: 4, AR197: 4, AR204: 4, AR177: 4, AR271: 4, AR217: 4, AR283: 4, AR291: 4, AR213: 4, AR169: 4, AR230: 4, AR224: 4, AR294: 4, AR290: 4, AR311: 4, AR277: 4, AR033: 4, AR195: 4, AR254: 4, AR308: 4, AR218: 4, AR191: 4, AR239: 4, AR223: 4, AR205: 4, AR236: 4, AR170: 4, AR295: 4, AR286: 4, AR203: 4, AR267: 4, AR212: 4, AR228: 4, AR219: 4, AR193: 4, AR192: 3, AR288: 3, AR266: 3, AR289: 3, AR255: 3, AR243: 3, AR172: 3, AR188: 3, AR227: 3, AR246: 3, AR232: 3, AR260: 2, AR168: 2, AR222: 2, AR250: 2, AR201: 2, AR225: 2, AR256: 2, AR190: 2, AR061: 2, AR055: 2, AR210: 2, AR211: 1, AR253: 1 H0422: 1 46 HHFHP90 534615 56  42-173 112 AR313: 36, AR089: 24, AR299: 23, AR242: 21, AR060: 18, AR196: 17, AR185: 16, AR316: 16, AR193: 16, AR096: 15, AR240: 14, AR192: 13, AR180: 12, AR300: 12, AR162: 12, AR039: 12, AR173: 12, AR175: 12, AR161: 12, AR104: 12, AR164: 12, AR163: 11, AR204: 11, AR165: 11, AR258: 11, AR197: 11, AR229: 11, AR166: 11, AR182: 11, AR277: 10, AR247: 10, AR199: 10, AR183: 10, AR198: 10, AR293: 10, AR262: 9, AR270: 9, AR235: 9, AR269: 9, AR179: 9, AR191: 9, AR238: 9, AR236: 9, AR178: 9, AR233: 9, AR234: 9, AR181: 9, AR195: 9, AR264: 9, AR257: 8, AR201: 8, AR053: 8, AR253: 8, AR312: 8, AR174: 8, AR285: 8, AR212: 8, AR245: 8, AR218: 8, AR296: 8, AR226: 8, AR207: 8, AR283: 7, AR282: 7, AR176: 7, AR243: 7, AR294: 7, AR287: 7, AR252: 7, AR254: 7, AR297: 7, AR271: 7, AR200: 7, AR275: 7, AR268: 7, AR213: 6, AR177: 6, AR255: 6, AR237: 6, AR231: 6, AR219: 6, AR188: 6, AR260: 6, AR267: 6, AR261: 6, AR228: 6, AR286: 6, AR295: 6, AR189: 5, AR290: 5, AR203: 5, AR291: 5, AR033: 5, AR274: 5, AR239: 5, AR288: 5, AR266: 5, AR205: 5, AR190: 4, AR309: 4, AR263: 4, AR230: 4, AR308: 4, AR227: 4, AR256: 4, AR289: 4, AR250: 3, AR221: 3, AR232: 3, AR246: 3, AR216: 3, AR217: 3, AR061: 2, AR211: 2, AR055: 2, AR311: 2, AR172: 2, AR210: 2, AR225: 2, AR222: 2, AR272: 1, AR169: 1, AR214: 1, AR224: 1, AR223: 1, AR215: 1 H0050: 2 and H0645: 1. 47 HLYCQ48 527757 57  58-252 113 Pro-42 to Lys-49. AR313: 35, AR196: 23, AR161: 19, AR173: 19, AR165: 19, AR242: 19, AR162: 18, AR164: 18, AR166: 18, AR089: 18, AR163: 18, AR247: 16, AR258: 16, AR300: 15, AR229: 15, AR183: 15, AR199: 15, AR240: 14, AR179: 14, AR096: 14, AR234: 14, AR175: 14, AR299: 13, AR178: 13, AR180: 13, AR185: 13, AR172: 13, AR262: 13, AR192: 12, AR181: 12, AR238: 12, AR257: 11, AR200: 11, AR174: 11, AR293: 11, AR191: 10, AR230: 10, AR312: 10, AR182: 10, AR270: 10, AR269: 10, AR177: 10, AR285: 10, AR233: 10, AR275: 10, AR193: 10, AR226: 10, AR296: 9, AR316: 9, AR203: 9, AR236: 9, AR218: 9, AR189: 9, AR260: 9, AR198: 9, AR060: 8, AR213: 8, AR277: 8, AR264: 8, AR297: 8, AR053: 8, AR039: 8, AR254: 8, AR231: 8, AR237: 8, AR195: 8, AR228: 8, AR268: 8, AR287: 7, AR294: 7, AR219: 7, AR286: 7, AR188: 7, AR204: 7, AR207: 7, AR261: 7, AR212: 6, AR033: 6, AR255: 6, AR239: 6, AR290: 6, AR282: 6, AR243: 6, AR176: 6, AR295: 6, AR227: 6, AR288: 5, AR274: 5, AR250: 5, AR256: 5, AR291: 5, AR309: 5, AR267: 5, AR104: 5, AR308: 5, AR245: 5, AR266: 5, AR201: 5, AR253: 4, AR289: 4, AR272: 4, AR190: 4, AR235: 4, AR232: 4, AR263: 4, AR197: 3, AR221: 3, AR205: 3, AR271: 3, AR216: 2, AR061: 2, AR225: 2, AR224: 1, AR311: 1, AR171: 1, AR223: 1, AR210: 1, AR211: 1 H0445: 1 48 HHLAB07 532602 58 108-317 114 AR225: 4, AR261: 4, AR282: 3, AR164: 3, AR172: 3, AR250: 3, AR264: 3, AR166: 3, AR163: 2, AR161: 2, AR207: 2, AR176: 2, AR204: 2, AR168: 2, AR217: 2, AR253: 2, AR270: 2, AR236: 2, AR181: 2, AR165: 2, AR300: 2, AR214: 2, AR266: 2, AR246: 2, AR263: 2, AR179: 2, AR183: 2, AR193: 1, AR228: 1, AR182: 1, AR267: 1, AR272: 1, AR233: 1, AR224: 1, AR221: 1, AR257: 1, AR178: 1, AR216: 1, AR287: 1, AR195: 1, AR277: 1, AR229: 1 S0116: 1, H0402: 1, T0091: 1 and S0052: 1. 49 HFOXE30 532120 59  38-199 115 AR217: 3, AR215: 3, AR169: 3, AR180: 3, AR176: 3, AR272: 3, AR172: 3, AR166: 2, AR225: 2, AR183: 2, AR309: 2, AR277: 2, AR195: 2, AR096: 2, AR274: 2, AR165: 2, AR246: 2, AR271: 2, AR230: 2, AR181: 2, AR164: 2, AR287: 2, AR264: 2, AR173: 2, AR039: 2, AR295: 1, AR224: 1, AR228: 1, AR282: 1, AR257: 1, AR269: 1, AR089: 1, AR192: 1, AR299: 1, AR175: 1, AR178: 1, AR222: 1, AR179: 1, AR060: 1, AR171: 1, AR289: 1, AR216: 1, AR296: 1, AR193: 1, AR311: 1, AR261: 1, AR182: 1, AR239: 1, AR205: 1, AR200: 1, AR236: 1, AR237: 1, AR238: 1 S0276: 1 50 HBJEL68 531924 60 109-231 116 Arg-32 to Gly-40. AR313: 12, AR161: 11, AR162: 11, AR163: 11, AR165: 10, AR164: 10, AR166: 10, AR176: 9, AR182: 9, AR089: 8, AR229: 8, AR196: 8, AR269: 8, AR181: 8, AR178: 7, AR257: 7, AR180: 7, AR238: 7, AR228: 7, AR060: 7, AR309: 7, AR275: 7, AR299: 7, AR247: 7, AR316: 7, AR233: 6, AR174: 6, AR173: 6, AR270: 6, AR183: 6, AR053: 6, AR293: 6, AR266: 6, AR200: 6, AR236: 6, AR226: 6, AR282: 6, AR175: 6, AR104: 6, AR240: 6, AR096: 6, AR300: 6, AR177: 6, AR191: 6, AR296: 6, AR189: 5, AR267: 5, AR274: 5, AR261: 5, AR264: 5, AR268: 5, AR185: 5, AR258: 5, AR262: 5, AR290: 5, AR237: 5, AR179: 5, AR199: 5, AR254: 5, AR312: 5, AR218: 5, AR277: 5, AR231: 5, AR239: 5, AR207: 5, AR234: 5, AR223: 5, AR168: 5, AR193: 5, AR039: 5, AR230: 4, AR235: 4, AR289: 4, AR204: 4, AR212: 4, AR198: 4, AR255: 4, AR285: 4, AR271: 4, AR227: 4, AR061: 4, AR297: 4, AR287: 4, AR308: 4, AR192: 4, AR288: 4, AR286: 4, AR242: 4, AR295: 4, AR203: 4, AR201: 4, AR188: 4, AR291: 4, AR225: 4, AR294: 4, AR195: 3, AR217: 3, AR055: 3, AR232: 3, AR219: 3, AR210: 3, AR214: 3, AR033: 3, AR190: 3, AR272: 3, AR283: 3, AR172: 3, AR224: 3, AR252: 3, AR245: 3, AR213: 3, AR256: 3, AR260: 3, AR216: 3, AR211: 2, AR250: 2, AR311: 2, AR222: 2, AR197: 2, AR263: 2, AR205: 1, AR170: 1, AR171: 1 S0222: 1 and H0318: 1. 50 HBJEL68 531873 65 111-191 121 51 HFOXA73 850699 61  25-180 117 AR264: 3, AR197: 3, AR274: 3, AR168: 2, AR291: 2, AR205: 2, AR283: 2, AR162: 2, AR163: 1, AR224: 1, AR161: 1, AR230: 1, AR240: 1, AR266: 1, AR190: 1, AR263: 1, AR191: 1, AR277: 1, AR178: 1, AR217: 1, AR257: 1, AR182: 1, AR295: 1 S0276: 1 51 HFOXA73 532079 66  15-68  122 52 HFIUR35 532062 62  42-254 118 Val-4 to Glu-9, AR176: 4, AR215: 3, Lys-42 to Lys-47. AR221: 3, AR313: 3, AR165: 3, AR170: 3, AR264: 3, AR168: 3, AR161: 3, AR164: 3, AR162: 3, AR163: 3, AR235: 2, AR053: 2, AR275: 2, AR309: 2, AR270: 2, AR089: 2, AR178: 2, AR254: 2, AR295: 2, AR262: 2, AR236: 2, AR242: 2, AR285: 2, AR277: 2, AR190: 2, AR166: 2, AR216: 2, AR257: 2, AR237: 2, AR171: 2, AR172: 2, AR185: 1, AR177: 1, AR243: 1, AR271: 1, AR312: 1, AR240: 1, AR191: 1, AR258: 1, AR183: 1, AR196: 1, AR222: 1, AR308: 1, AR247: 1, AR316: 1, AR300: 1, AR299: 1, AR228: 1, AR189: 1, AR234: 1, AR272: 1, AR104: 1, AR180: 1, AR268: 1, AR294: 1, AR175: 1, AR233: 1, AR229: 1, AR179: 1, AR269: 1 L0748: 1, L0608: 1 and S0196: 1. 53 HFPDE86 527728 63 300-350 119 AR313: 48, AR229: 26, AR300: 26, AR173: 25, AR096: 23, AR258: 23, AR299: 22, AR164: 22, AR257: 22, AR165: 22, AR161: 21, AR162: 21, AR262: 21, AR180: 20, AR163: 20, AR247: 20, AR166: 20, AR175: 20, AR178: 19, AR183: 18, AR179: 18, AR181: 18, AR218: 18, AR270: 18, AR234: 17, AR226: 17, AR196: 17, AR089: 17, AR269: 17, AR240: 16, AR238: 16, AR185: 15, AR199: 14, AR174: 14, AR182: 14, AR191: 14, AR177: 13, AR268: 13, AR233: 13, AR293: 13, AR231: 13, AR296: 13, AR219: 13, AR312: 12, AR230: 12, AR189: 12, AR237: 12, AR316: 12, AR203: 12, AR260: 11, AR255: 11, AR236: 11, AR264: 11, AR176: 11, AR261: 10, AR267: 10, AR282: 10, AR060: 10, AR275: 10, AR053: 10, AR228: 10, AR266: 10, AR286: 9, AR285: 9, AR239: 9, AR188: 9, AR242: 9, AR200: 9, AR033: 8, AR297: 8, AR274: 8, AR256: 8, AR294: 8, AR193: 8, AR212: 7, AR277: 7, AR227: 7, AR309: 7, AR290: 7, AR213: 7, AR192: 6, AR190: 6, AR039: 6, AR308: 6, AR287: 6, AR232: 6, AR263: 6, AR250: 6, AR104: 6, AR295: 6, AR289: 6, AR291: 6, AR169: 5, AR288: 5, AR210: 5, AR272: 5, AR243: 5, AR253: 5, AR195: 4, AR211: 4, AR170: 4, AR283: 4, AR204: 4, AR311: 4, AR172: 4, AR235: 4, AR254: 3, AR225: 3, AR197: 3, AR205: 3, AR245: 3, AR252: 3, AR061: 3, AR201: 3, AR055: 3, AR271: 3, AR216: 2, AR217: 2, AR214: 2, AR246: 2, AR223: 2, AR207: 2, AR168: 2, AR171: 2, AR224: 1, AR198: 1 S0222: 1 and L0682: 1.

[0462] TABLE 1C SEQ ID cDNA NO: CONTIG SEQ ID EXON Clone ID X ID: BAC ID: A NO: B From-To HSIDU19 11 520372 AL391194 220  1-83 166-317  639-1142 1280-1372 2309-2753 HSIDU19 11 520372 AL391194 221  1-262 HSIDU19 11 520372 AL391194 222  1-768 HTEIL66 13 520125 AC067727 223  1-507 2558-2661 2869-3237 HSAXB32 16 520470 AC002369 224  1-586 2559-2651 3329-3426 3756-5088 HSAXB32 16 520470 AC011957 225  1-126 HSAXB32 16 520470 AC016655 226   1-1025 HSAXB32 16 520470 AC010419 227   1-1024 HSAXB32 16 520470 AC027536 228  1-119 HSAXB32 16 520470 AC018548 229   1-1025 HSAXB32 16 520470 AP001973 230  1-102 HSAXB32 16 520470 AC002369 231  1-228 HSAXB32 16 520470 AC016655 232  1-729 HSAXB32 16 520470 AC016655 233  1-244 HSAXB32 16 520470 AC010419 234  1-732 HSAXB32 16 520470 AC010419 235  1-244 HSAXB32 16 520470 AC018548 236  1-730 HSAXB32 16 520470 AC018548 237  1-244 HPVAB94 18 526749 AF165147 238   1-1087 HPVAB94 18 526749 AF131217 239   1-1087 HSAXB81 19 520471 AL353771 240   1-5816 6416-7049 8127-8227 8937-8991 10861-10999 11231-11539 12714-12960 14741-15620 15735-16103 16263-16776 18121-18258 19724-20346 20585-21454 22251-22431 22675-22937 23080-23487 23555-23944 24043-26493 HSAXB81 19 520471 AC019213 241  1-880 HSAXB81 19 520471 AL354721 242  1-880 HSAXB81 19 520471 AL353771 243  1-437 HSAXB81 19 520471 AL353771 244  1-339 HSAXB81 19 520471 AC019213 245   1-5816 6416-6688 8132-8232 8942-8996 10872-11005 11237-11545 12720-12966 HSAXB81 19 520471 AC019213 246  1-369 HSAXB81 19 520471 AL354721 247  1-369 HSAYC21 20 526188 AC019279 248  1-637 HSAYC21 20 526188 AC011097 249  1-647 HSAYC21 20 526188 AC011097 250  1-324 HSAYC21 20 526188 AC011097 251  1-281 HSLCU73 21 520237 AL138784 252  1-456 1098-1456 1870-2167 2841-2938 3718-4105 4803-5382 5830-5976 6102-6299 6733-6832 7071-7843 8487-9355  9898-10417 10629-11625 11647-11696 11704-11741 11813-12296 12489-13208 15576-15724 18189-18478 18985-19068 21115-21267 26441-26769 26782-27289 27493-27991 29015-29282 29428-29482 29508-29933 29940-30050 30185-31063 31143-32602 33715-34300 34847-35835 36406-36719 36786-36929 37366-37814 38984-39108 40994-41111 41559-43991 HSLCU73 21 520237 AL138784 253  1-450 HSSFZ70 22 526499 AP002833 254  1-586 HSSFZ70 22 526499 AP000839 255  1-585 HSSFZ70 22 526499 AP002833 256  1-639 HSSFZ70 22 526499 AP000839 257  1-639 HTEIP36 23 520468 AC037454 258  1-37 2996-3803 HSAVP17 26 519846 AC010633 259   1-1273 HSAVP17 26 519846 AC010633 260  1-484 HSAVP17 26 519846 AC010633 261  1-140 HSIEA14 27 520387 AL133519 262  1-368 1447-1751 1777-2025 3643-4256 6078-6205 6273-6840 8298-8430 10016-10172 16541-16665 17747-17893 18161-18547 18862-19340 19799-20268 23067-23841 23848-23950 26005-26779 28885-28998 29777-29973 HSIEA14 27 520387 AL109655 263  1-775 HSIEA14 27 520387 AL133519 264  1-353  402-1224 HSNAQ47 28 847453 AC024468 265  1-835 HSNAQ47 28 847453 AL162612 266   1-3929 HSNAQ47 28 847453 AC018593 267   1-3928 HSNAQ47 28 847453 AC025992 268   1-3928 HSNAQ47 28 847453 AC024468 269  1-269 2419-2560 3305-3786 4474-5582 5736-6454 6554-8813 9717-9903 HSNAQ47 28 847453 AL162612 270  1-379 HSNAQ47 28 847453 AC018593 271  1-379 HSNAQ47 28 847453 AC025992 272  1-379 HODDN65 29 520348 AC073052 273  1-753 HODDN65 29 520348 AC073052 274  1-101 HPEAD79 30 520202 AL158846 275  1-811 HPEAD79 30 520202 AL158846 276  1-98 HPEAD79 30 520202 AL158846 277  1-101 HRDED19 31 526019 AC021221 278  1-506 HRDED19 31 526019 AC040946 279  1-506 HSAYS89 32 526621 AC069204 280  1-624 HSAYS89 32 526621 AC011319 281  1-624 HSAYS89 32 526621 AC069204 282  1-146 HSVAM10 34 520328 AC021164 283  1-363 HSVAM10 34 520328 AC021164 284  1-164 HSVAM10 34 520328 AC021164 285  1-177 HSNBN57 35 526721 AC007263 286  1-629 HSNBN57 35 526721 AL157736 287  1-629 HSNBN57 35 526721 AL357093 288  1-629 HSVBD22 36 520557 AL138996 289  1-600 HSVBD22 36 520557 AL138996 290  1-311 HSAWA27 37 520302 AL358073 291  1-646 HSAWA27 37 520302 AL358353 292  1-646 HSAWA27 37 520302 AC022243 293  1-646 HSAWA27 37 520302 AL358073 294  1-138 HSAWA27 37 520302 AC022243 295  1-138 HSFAH43 38 520399 Z99758 296  1-432 1573-1657 2425-2702 2830-3126 3331-3848 4530-8774  8939-10624 26471-32522 HSFAH43 38 520399 Z95400 297   1-2805 2807-3714 HSFAH43 38 520399 Z92542 298  1-728 3448-3710 6061-6275 HSFAH43 38 520399 U69729 299   1-2395 HSFAH43 38 520399 AP001821 300   1-1750 HSFAH43 38 520399 AP000068 301   1-6128 HSFAH43 38 520399 AL391071 302  1-29 2979-3837 HSFAH43 38 520399 AL365475 303   1-5542  9722-10059 12469-12825 17209-18808 23300-23420 28096-28311 33950-34377 36732-36948 45973-46071 46680-49375 HSFAH43 38 520399 AL356802 304   1-6186 HSFAH43 38 520399 AL355052 305   1-6182 HSFAH43 38 520399 AL354833 306  1-999 HSFAH43 38 520399 AL354751 307   1-6172 HSFAH43 38 520399 AL353812 308   1-6038 HSFAH43 38 520399 AL162390 309  1-442 3536-4070  8479-10006 11103-11289 16064-16381 18580-19340 21066-21597 HSFAH43 38 520399 AL157915 310   1-1074 HSFAH43 38 520399 AL138810 311   1-2603 3747-3810 4195-4655 8673-9491 12027-15126 18182-18479 22522-22762 25837-25984 28990-29105 29310-29482 31547-31981 37753-38223 42637-45774 HSFAH43 38 520399 AL135978 312   1-1943 HSFAH43 38 520399 AL133381 313   1-3135 HSFAH43 38 520399 AL133370 314   1-1073 HSFAH43 38 520399 AL133304 315   1-1241 HSFAH43 38 520399 AL132826 316   1-5842 HSFAH43 38 520399 AL121879 317   1-3114 HSFAH43 38 520399 AL110502 318  1-386  396-2931 HSFAH43 38 520399 AL109628 319   1-1450 HSFAH43 38 520399 AL079303 320  1-27  49-784 HSFAH43 38 520399 AL078644 321  1-702  835-6171 HSFAH43 38 520399 AL049875 322   1-1845 HSFAH43 38 520399 AL035700 323   1-2442 HSFAH43 38 520399 AL035689 324   1-1474 HSFAH43 38 520399 AL034408 325  1-337 1909-5834 HSFAH43 38 520399 AL033522 326   1-2391 HSFAH43 38 520399 AL033403 327  1-444  466-2420 HSFAH43 38 520399 AL031650 328   1-6176 10689-11002 HSFAH43 38 520399 AL031387 329   1-5310 HSFAH43 38 520399 AL024507 330   1-6096 HSFAH43 38 520399 AL022395 331  1-639 4341-6175 HSFAH43 38 520399 AL022394 332   1-1723 HSFAH43 38 520399 AL022165 333  1-300 1441-7576 HSFAH43 38 520399 AL022069 334   1-4180 HSFAH43 38 520399 AL021408 335   1-1600 HSFAH43 38 520399 AF207550 336   1-1946 HSFAH43 38 520399 AF130343 337   1-2009 HSFAH43 38 520399 AF027390 338  1-799 HSFAH43 38 520399 AF011889 339  1-127  131-6009 HSFAH43 38 520399 AF001905 340  1-895 HSFAH43 38 520399 AC022078 341   1-6035 12528-12716 12739-13728 17358-17923 18326-18548 20169-20267 20945-21314 23341-24951 27225-27416 35928-36115 36448-36827 39241-39566 40073-42103 42112-42317 42448-44311 HSFAH43 38 520399 AC011327 342   1-4926 7254-7384 HSFAH43 38 520399 AC009948 343   1-2807 2928-3084 3825-4520 4618-4772 5297-5601 5669-7057 7150-8193 8825-8961 9412-9822 HSFAH43 38 520399 AC009501 344  1-116 4875-5137 5347-6009 6286-6690 6808-7890 9458-9518 14225-14447 28608-28803 30489-30789 35053-39752 45421-45698 HSFAH43 38 520399 AC009289 345   1-6032 6037-6158  7763-11661 HSFAH43 38 520399 AC008583 346   1-2169 HSFAH43 38 520399 AC008134 347   1-5956 HSFAH43 38 520399 AC008041 348   1-3014 HSFAH43 38 520399 AC007744 349   1-1223 HSFAH43 38 520399 AC007743 350   1-2579 HSFAH43 38 520399 AC007570 351  1-441  848-1703 2937-3091 4107-4235 5420-5837 6762-6878 7258-7362 7851-8552 11012-11366 11683-11945 11992-12100 14355-15033 16562-16726 18157-18321 18716-18981 21960-22123 23221-23312 24248-24333 HSFAH43 38 520399 AC007436 352  1-710 HSFAH43 38 520399 AC007358 353  1-916 HSFAH43 38 520399 AC007276 354   1-1511 2538-2927 4237-4537 5726-6097 6190-6923 8701-8766 HSFAH43 38 520399 AC007000 355   1-2015 HSFAH43 38 520399 AC006984 356  1-832 HSFAH43 38 520399 AC006556 357   1-5944 8943-9234 12643-13118 15612-15759 15811-15938 15947-17554 24155-24455 34509-36783 HSFAH43 38 520399 AC006313 358   1-1650 HSFAH43 38 520399 AC006226 359  1-211 1700-3957 4145-6149 HSFAH43 38 520399 AC006195 360   1-5323 HSFAH43 38 520399 AC006144 361  1-865 HSFAH43 38 520399 AC006084 362   1-6054 HSFAH43 38 520399 AC005740 363   1-3402 HSFAH43 38 520399 AC005166 364   1-6073 7526-7932 7938-8353 11867-12326 13590-14009 19843-20521 20684-21147 23387-25220 HSFAH43 38 520399 AC005164 365   1-6153 HSFAH43 38 520399 AC005146 366   1-2558 HSFAH43 38 520399 AC005090 367   1-6048 21583-23065 HSFAH43 38 520399 AC005023 368   1-4659 5290-6014 HSFAH43 38 520399 AC003099 369   1-1750 HSFAH43 38 520399 AC003075 370  1-841 HSFAH43 38 520399 AC002379 371   1-2312 HSFAH43 38 520399 AC002060 372   1-1446 HSFAH43 38 520399 AC000120 373   1-6147 HSFAH43 38 520399 AB045359 374   1-1022 HSFAH43 38 520399 AC023555 375   1-6203 HSFAH43 38 520399 AC011739 376   1-2587 HSFAH43 38 520399 AL159175 377   1-1638 HSFAH43 38 520399 AL158158 378   1-1176  4708-10789 HSFAH43 38 520399 AC021201 379   1-1440 3030-3062 HSFAH43 38 520399 AC011768 380   1-1733 1937-2229 2247-2452 2914-3752 4277-4330 4469-6688 7268-7948 HSFAH43 38 520399 AL359754 381  1-248 1315-1839 2740-6257 HSFAH43 38 520399 AL355474 382  1-357 1494-1815 2463-5308 5329-8041 HSFAH43 38 520399 AL139136 383   1-2735 HSFAH43 38 520399 AC012207 384   1-6160 HSFAH43 38 520399 AC009266 385   1-5516 11501-13845 HSFAH43 38 520399 AC005000 386  1-772 1063-1418 1837-2098 2118-2246 2664-2705 2758-2890 3334-5823 HSFAH43 38 520399 AL355305 387   1-1431 3215-3361 4470-4666 4833-4908 5442-5931 6349-6723 7136-8144 8628-8720  9983-10102 11507-11655 13693-13817 13820-15838 16993-17177 18554-18715 HSFAH43 38 520399 AL133385 388   1-3135 HSFAH43 38 520399 AC012447 389   1-2238 HSFAH43 38 520399 AC010192 390   1-1031 HSFAH43 38 520399 AC004958 391   1-6152 HSFAH43 38 520399 AP000409 392   1-2292 2296-3173 HSFAH43 38 520399 AL356259 393  1-844 HSFAH43 38 520399 AL138814 394  1-217  401-3497 5172-5396 7866-8155 8596-9620 10961-11122 HSFAH43 38 520399 AC025555 395   1-1656 HSFAH43 38 520399 AC025508 396   1-6052 HSFAH43 38 520399 AC022845 397  1-792 HSFAH43 38 520399 AC012321 398  1-312 532-920 1441-2015 2738-4897 HSFAH43 38 520399 AC012036 399   1-1435 HSFAH43 38 520399 AL356513 400   1-4544 4599-4917 HSFAH43 38 520399 AL121933 401   1-1249 HSFAH43 38 520399 AC069443 402  1-153 4951-5981 HSFAH43 38 520399 AC068139 403   1-1702 HSFAH43 38 520399 AC011435 404   1-6054 HSFAH43 38 520399 AC011153 405   1-2044 HSFAH43 38 520399 AC006357 406   1-6191 HSFAH43 38 520399 AC011401 407   1-3406 HSFAH43 38 520399 AC023965 408   1-1086 HSFAH43 38 520399 AL359271 409   1-1840 HSFAH43 38 520399 AL355075 410  1-716 HSFAH43 38 520399 AL109804 411  1-811 HSFAH43 38 520399 AC023109 412  1-447  479-3690 3704-4456 HSFAH43 38 520399 AC012600 413   1-1190 HSFAH43 38 520399 AC027552 414   1-1081 HSFAH43 38 520399 AP001645 415   1-2650 HSFAH43 38 520399 AP001567 416   1-6410 HSFAH43 38 520399 AP001562 417   1-4260 HSFAH43 38 520399 AP000598 418  1-838 HSFAH43 38 520399 AL356982 419   1-2358 HSFAH43 38 520399 AL354708 420  1-481  501-1170 HSFAH43 38 520399 AL157899 421  1-738  792-6970 HSFAH43 38 520399 AL157762 422   1-2317 2972-3275 HSFAH43 38 520399 AC024947 423   1-2765 HSFAH43 38 520399 AC010210 424   1-6175 HSFAH43 38 520399 AC007849 425   1-6052 HSFAH43 38 520399 AC007338 426   1-1055 HSFAH43 38 520399 AC022122 427   1-2439 HSFAH43 38 520399 AL031601 428   1-3441 HSFAH43 38 520399 AC058811 429  1-176  769-4422 4453-4885 4939-5853 HSFAH43 38 520399 AC022194 430  1-667 874-966 2107-7838 HSFAH43 38 520399 AC068393 431   1-3748 HSFAH43 38 520399 AC025624 432   1-2446 HSFAH43 38 520399 AC074250 433  1-59 248-396 5576-5669 7524-7687 8145-8533 8571-8834 9397-9870 11221-11322 11562-11960 12482-13656 16780-16900 17004-17101 17108-17489 17592-18932 HSFAH43 38 520399 AC023412 434  1-609  774-4893 4947-5863 HSFAH43 38 520399 AC027116 435   1-1733 1937-2229 2247-2452 2914-3752 4277-4330 4469-6688 7268-7948 HSFAH43 38 520399 AC026494 436   1-1041 1050-3506 HSFAH43 38 520399 AC073118 437   1-2077 HSFAH43 38 520399 AP001321 438   1-6041 HSFAH43 38 520399 AP000974 439  1-792 HSFAH43 38 520399 AP000445 440   1-4775 HSFAH43 38 520399 AL355489 441   1-2350 HSFAH43 38 520399 AL161645 442   1-1620 HSFAH43 38 520399 AL139109 443  1-198  203-6067 HSFAH43 38 520399 AL138682 444  1-217  401-3497 5172-5396 7866-8155 8602-9620 10961-11122 HSFAH43 38 520399 AL133124 445   1-2174 HSFAH43 38 520399 AL031726 446  1-751 HSFAH43 38 520399 AC078802 447   1-6052 HSFAH43 38 520399 AC073487 448  1-587 2146-3842 3989-4201 5499-5529 6178-6728 7177-7679 8088-9978 10716-10977 10981-11359 11459-11652 11709-12255 12352-12457 12485-12662 12775-12853 12946-13797 13854-13972 14616-14764 15230-15403 15550-15610 15784-15879 16027-16157 16273-16359 16716-16906 17040-17252 18205-18727 18898-19487 20516-20784 21205-21286 21386-21515 25809-26225 HSFAH43 38 520399 AC069525 449   1-5002 HSFAH43 38 520399 AC063920 450  1-93 143-454 1232-6963 HSFAH43 38 520399 AC021652 451  1-667 874-966 2107-7838 HSFAH43 38 520399 AC021329 452   1-1733 1937-2229 2257-2450 2914-3752 4277-4330 4728-4851 HSFAH43 38 520399 AC020656 453   1-2070 HSFAH43 38 520399 AC018845 454   1-1055 HSFAH43 38 520399 AC018840 455   1-4959 7250-7380 HSFAH43 38 520399 AC018494 456  1-762 HSFAH43 38 520399 AC016119 457  1-951 HSFAH43 38 520399 AC011432 458   1-1032 4529-5194 5642-5695 17014-22913 HSFAH43 38 520399 AC010798 459   1-1440 1543-8503 HSFAH43 38 520399 AC007623 460  1-441  848-1703 2937-3091 4107-4235 5420-5837 6762-6878 7258-7362 7851-8552 11012-11366 11683-11945 11992-12100 14355-15033 16562-16726 18157-18321 18716-18981 21960-22123 23221-23312 24248-24333 HSFAH43 38 520399 AC007345 461  1-32  142-6020 HSFAH43 38 520399 AC015521 462   1-2151 HSFAH43 38 520399 AC069153 463   1-1431 3215-3361 4470-4666 4833-4908 5442-5931 6349-6723 7136-8144 8628-8720  9983-10102 13246-13371 13390-13464 13683-13807 13810-15828 16983-17167 18544-18705 HSFAH43 38 520399 AC015464 464   1-3481 HSFAH43 38 520399 AC010736 465   1-2077 HSFAH43 38 520399 AC025802 466   1-2446 HSFAH43 38 520399 AC018568 467   1-1645 HSFAH43 38 520399 AC074245 468  1-640  816-1328 1629-3899 4841-4907 6138-6199 6282-6407 HSFAH43 38 520399 AC011823 469   1-2288 2442-3104 4477-4593 13213-13374 13769-13977 15164-15429 15575-15735 15834-15933 16814-18957 19984-20104 20707-20794 22341-22556 24065-24247 24773-24898 25233-25574 26907-27007 28468-29216 29538-30106 30118-30602 30656-31302 31349-31375 31381-31559 36563-41903 41914-42326 42427-42595 HSFAH43 38 520399 AC011123 470   1-4261 HSFAH43 38 520399 AC013708 471   1-1912 1930-2850 3505-3808 HSFAH43 38 520399 AC037472 472   1-1127 HSFAH43 38 520399 AC011086 473  1-865 HSFAH43 38 520399 AL136327 474   1-1175 HSFAH43 38 520399 AC021850 475   1-2657 HSFAH43 38 520399 Z92542 476  1-230 HSFAH43 38 520399 U69729 477  1-486 HSFAH43 38 520399 U69729 478  1-141 HSFAH43 38 520399 AL391071 479  1-228 HSFAH43 38 520399 AL354833 480  1-457 HSFAH43 38 520399 AL354751 481  1-349 HSFAH43 38 520399 AL354751 482  1-274 HSFAH43 38 520399 AL162390 483  1-187 HSFAH43 38 520399 AL162390 484  1-202 HSFAH43 38 520399 AL157915 485  1-602 HSFAH43 38 520399 AL157915 486  1-543 HSFAH43 38 520399 AL138810 487  1-716 HSFAH43 38 520399 AL133370 488  1-602 HSFAH43 38 520399 AL133370 489  1-543 HSFAH43 38 520399 AL132826 490  1-102 HSFAH43 38 520399 AL110502 491  1-529 HSFAH43 38 520399 AL078644 492  1-326 HSFAH43 38 520399 AL035689 493  1-105 HSFAH43 38 520399 AL035689 494  1-139 HSFAH43 38 520399 AL034408 495  1-519 HSFAH43 38 520399 AL033403 496  1-130 820-951 1765-1857 5765-5858 6925-7187 8041-8110 8229-8343 9072-9146 9399-9769 10180-10631 11051-11151 HSFAH43 38 520399 AL024507 497  1-134 HSFAH43 38 520399 AL022395 498   1-1691 1822-2256 3102-3417 3512-4108 4358-4629 4923-6465 6879-7193 12019-12091 14772-14872 17599-18179 22036-22216 22839-23187 28010-28356 29023-29363 32379-32844 HSFAH43 38 520399 AL022395 499  1-514 HSFAH43 38 520399 AL022394 500  1-132 HSFAH43 38 520399 AL022165 501  1-172 5426-5577 7890-8007 8100-8182 10387-10472 HSFAH43 38 520399 AL022165 502  1-424 HSFAH43 38 520399 AL022069 503  1-170 HSFAH43 38 520399 AF207550 504  1-54 170-273 5331-5457 5684-5983 5992-6112 6853-6948 7283-7498 7631-7747 8055-8170 8851-8931 9035-9130 9252-9308 9410-9511  9831-10176 11703-12423 13776-14987 15189-15458 15854-16422 HSFAH43 38 520399 AF130343 505  1-126 5612-5752 HSFAH43 38 520399 AC022078 506  1-828 HSFAH43 38 520399 AC011327 507  1-433 HSFAH43 38 520399 AC009948 508  1-298 HSFAH43 38 520399 AC009948 509  1-651 HSFAH43 38 520399 AC008583 510   1-1146 HSFAH43 38 520399 AC008134 511  1-174 HSFAH43 38 520399 AC008041 512  1-392 HSFAH43 38 520399 AC007570 513  1-167 1801-1865 4645-4745 6582-7129 8325-8394 11657-11736 11991-12093 12184-12269 12394-12473 14154-14418 14760-15011 15741-18861 HSFAH43 38 520399 AC007000 514  1-387 HSFAH43 38 520399 AC007000 515  1-117 HSFAH43 38 520399 AC006984 516  1-374 HSFAH43 38 520399 AC006144 517  1-280 HSFAH43 38 520399 AC006084 518  1-165 HSFAH43 38 520399 AC006084 519  1-544 HSFAH43 38 520399 AC005740 520  1-140 HSFAH43 38 520399 AC002379 521  1-370 HSFAH43 38 520399 AC002379 522  1-465 HSFAH43 38 520399 AC002060 523  1-420 HSFAH43 38 520399 AC000120 524  1-247 HSFAH43 38 520399 AL158158 525  1-617 1915-4051 4392-4708 6818-7286 HSFAH43 38 520399 AC011768 526  1-457 593-900  922-1022 HSFAH43 38 520399 AL359754 527  1-227 HSFAH43 38 520399 AC012207 528  1-515 HSFAH43 38 520399 AL355305 529  1-327 HSFAH43 38 520399 AC012447 530  1-88 HSFAH43 38 520399 AC010192 531  1-287 HSFAH43 38 520399 AP000409 532  1-183 1203-1305 3252-3431 3478-5013 HSFAH43 38 520399 AL138814 533  1-543 HSFAH43 38 520399 AC025508 534  1-189 HSFAH43 38 520399 AC022845 535  1-419 HSFAH43 38 520399 AC012036 536  1-297 HSFAH43 38 520399 AL356513 537  1-296 1293-1827 1881-2275 2436-2731 HSFAH43 38 520399 AL121933 538  1-100 HSFAH43 38 520399 AC069443 539  1-84 HSFAH43 38 520399 AC069443 540  1-287 HSFAH43 38 520399 AC011435 541  1-544 HSFAH43 38 520399 AC006357 542  1-360 HSFAH43 38 520399 AC011401 543  1-142 HSFAH43 38 520399 AL359271 544  1-331 HSFAH43 38 520399 AL355075 545  1-551 HSFAH43 38 520399 AC012600 546  1-165 HSFAH43 38 520399 AC012600 547  1-285 HSFAH43 38 520399 AC027552 548  1-145 HSFAH43 38 520399 AP001567 549   1-1859 HSFAH43 38 520399 AP001562 550  1-381 HSFAH43 38 520399 AP001562 551  1-663 HSFAH43 38 520399 AP000598 552  1-965 1032-1733 1937-2229 2257-2448 HSFAH43 38 520399 AL354708 553  1-389 HSFAH43 38 520399 AL354708 554  1-606 HSFAH43 38 520399 AL157899 555  1-288 HSFAH43 38 520399 AL157762 556  1-366 HSFAH43 38 520399 AC010210 557  1-373 HSFAH43 38 520399 AC007849 558  1-89 HSFAH43 38 520399 AC007338 559  1-117 HSFAH43 38 520399 AL031601 560  1-417 HSFAH43 38 520399 AC022194 561  1-379 HSFAH43 38 520399 AC022194 562  1-151 HSFAH43 38 520399 AC025624 563  1-531 HSFAH43 38 520399 AC025624 564  1-503 HSFAH43 38 520399 AC027116 565  1-59  76-532 668-975  995-1151 2499-2635 HSFAH43 38 520399 AC026494 566  1-459 HSFAH43 38 520399 AC073118 567  1-553 HSFAH43 38 520399 AP000974 568  1-419 HSFAH43 38 520399 AP000445 569  1-331 HSFAH43 38 520399 AP000445 570  1-206 HSFAH43 38 520399 AL161645 571  1-410 HSFAH43 38 520399 AL161645 572  1-868 HSFAH43 38 520399 AL138682 573  1-543 HSFAH43 38 520399 AL133124 574   1-1512 HSFAH43 38 520399 AL133124 575  1-324 HSFAH43 38 520399 AL031726 576   1-3011 3222-3912 HSFAH43 38 520399 AC078802 577  1-89 HSFAH43 38 520399 AC073487 578  1-259 HSFAH43 38 520399 AC069525 579  1-179 HSFAH43 38 520399 AC063920 580  1-378 HSFAH43 38 520399 AC063920 581  1-386 HSFAH43 38 520399 AC021652 582  1-379 HSFAH43 38 520399 AC021652 583  1-151 HSFAH43 38 520399 AC018845 584  1-117 HSFAH43 38 520399 AC018840 585  1-433 HSFAH43 38 520399 AC016119 586  1-449 HSFAH43 38 520399 AC011432 587  1-523 HSFAH43 38 520399 AC011432 588  1-116 HSFAH43 38 520399 AC007623 589  1-167 1804-1865 4649-4745 HSFAH43 38 520399 AC007345 590  1-114 HSFAH43 38 520399 AC015521 591   1-1146 HSFAH43 38 520399 AC069153 592  1-327 HSFAH43 38 520399 AC010736 593  1-553 HSFAH43 38 520399 AC025802 594  1-503 HSFAH43 38 520399 AC025802 595  1-531 HSFAH43 38 520399 AC018568 596  1-239 HSFAH43 38 520399 AC074245 597  1-158 HSFAH43 38 520399 AC011823 598  1-120 175-422 779-1165 HSFAH43 38 520399 AC011123 599  1-663 HSFAH43 38 520399 AC011123 600  1-381 HSFAH43 38 520399 AC013708 601  1-366 HSFAH43 38 520399 AC037472 602  1-108 HSFAH43 38 520399 AL136327 603  1-399 HSPAA60 39 526447 Z98044 604  1-462 HNGBX63 43 532615 AL137077 605  1-514 HE2AG50 44 532595 AC026052 606  1-451  830-2549 HE2AG50 44 532595 AL357314 607  1-451  830-2548 HE2AG50 44 532595 AC009337 608  1-228  696-1889 HE2AG50 44 532595 AL357314 609  1-84 HCUIN80 45 534128 AC008465 610  1-813 HADCL29 46 532056 AC025153 611  1-998 HADCL29 46 532056 AC025153 612  1-134 HADCL29 46 532056 AC025153 613  1-88 HAPPS89 47 532135 AC007459 614  1-242 HAPPS89 47 532135 AC002369 615  1-586 2559-2651 3329-3426 3756-5088 HAPPS89 47 532135 AL390917 616  1-181 HAPPS89 47 532135 AC021401 617  1-319 HAPPS89 47 532135 AC013758 618  1-316 HAPPS89 47 532135 AC073909 619  1-286 HAPPS89 47 532135 AL354657 620  1-305 HAPPS89 47 532135 AL161634 621  1-300 HAPPS89 47 532135 AC068196 622  1-316 HAPPS89 47 532135 AC021901 623  1-283 HAPPS89 47 532135 AC009407 624  1-450 3345-4200 HAPPS89 47 532135 AC021921 625  1-170 HAPPS89 47 532135 AC023968 626  1-306 HAPPS89 47 532135 AC022051 627  1-294 HAPPS89 47 532135 AC019092 628  1-184 HAPPS89 47 532135 AC011233 629  1-258 HAPPS89 47 532135 AC060817 630  1-299 HAPPS89 47 532135 AL353139 631  1-316 HAPPS89 47 532135 AC024337 632  1-235 HAPPS89 47 532135 AL358492 633  1-44 HAPPS89 47 532135 AC055788 634  1-170 HAPPS89 47 532135 AC023672 635  1-196 HAPPS89 47 532135 AC009899 636  1-175 HAPPS89 47 532135 AC009095 637  1-196 HAPPS89 47 532135 AP001929 638  1-301 HAPPS89 47 532135 AC012461 639  1-207 HAPPS89 47 532135 AC027772 640  1-189 HAPPS89 47 532135 AC027408 641  1-207 HAPPS89 47 532135 AC027584 642  1-162 HAPPS89 47 532135 AC016538 643  1-301 HAPPS89 47 532135 AP001992 644  1-301 HAPPS89 47 532135 AC067907 645  1-291 HAPPS89 47 532135 AC023309 646  1-193 HAPPS89 47 532135 AC041025 647  1-206 3881-3991 HAPPS89 47 532135 AC073446 648  1-140 HAPPS89 47 532135 AC026936 649  1-280 HAPPS89 47 532135 AC024475 650  1-187 HAPPS89 47 532135 AC067779 651  1-246 HAPPS89 47 532135 AC009858 652  1-39 HAPPS89 47 532135 AC021164 653  1-315 3876-3905 HAPPS89 47 532135 AC068682 654  1-153 HAPPS89 47 532135 AC034137 655  1-202 HAPPS89 47 532135 AL356423 656  1-203 HAPPS89 47 532135 AL157775 657  1-280 HAPPS89 47 532135 AC068755 658  1-190 HAPPS89 47 532135 AC034243 659  1-312 2334-2364 HAPPS89 47 532135 AC021212 660  1-304 HAPPS89 47 532135 AC015604 661  1-261 HAPPS89 47 532135 AC036208 662  1-450 3345-4200 HAPPS89 47 532135 AC027234 663  1-318 HAPPS89 47 532135 AC068519 664  1-312 HAPPS89 47 532135 AC016319 665  1-191 HAPPS89 47 532135 AC011036 666  1-193 HAPPS89 47 532135 AC010853 667  1-195 HAPPS89 47 532135 AC055861 668  1-281 HAPPS89 47 532135 AC025869 669  1-323 HAPPS89 47 532135 AC073849 670  1-309 HAPPS89 47 532135 AC072051 671  1-310 HAPPS89 47 532135 AC025975 672  1-136 HAPPS89 47 532135 AC015587 673  1-273 HAPPS89 47 532135 AL353577 674  1-279 HAPPS89 47 532135 AC010073 675  1-243 HAPPS89 47 532135 AL353659 676  1-202 HAPPS89 47 532135 AC068289 677  1-192 HAPPS89 47 532135 AC022795 678  1-300 HAPPS89 47 532135 AL355975 679  1-322 HAPPS89 47 532135 AC023864 680  1-142 HAPPS89 47 532135 AL354696 681  1-181 HAPPS89 47 532135 AC073219 682  1-123 HAPPS89 47 532135 AL139131 683  1-214 HAPPS89 47 532135 AC022766 684  1-318 HAPPS89 47 532135 AC027442 685  1-307 HAPPS89 47 532135 AC020873 686  1-306 HAPPS89 47 532135 AC002369 687  1-228 HAPPS89 47 532135 AL390917 688  1-131 HAPPS89 47 532135 AC068196 689  1-214 HAPPS89 47 532135 AC009407 690  1-98 HAPPS89 47 532135 AC019092 691  1-268 HAPPS89 47 532135 AC027584 692  1-368 HAPPS89 47 532135 AC073446 693  1-52 2626-2925 HAPPS89 47 532135 AC067779 694   1-1558 1589-4706 HAPPS89 47 532135 AC009858 695  1-212 HAPPS89 47 532135 AC036208 696  1-98 HAPPS89 47 532135 AC023864 697   1-1485 1590-4704 HAPPS89 47 532135 AC020873 698  1-126 HFGAH44 48 533132 AC023120 699   1-1768 HFGAH44 48 533132 AC023120 700  1-127 HFGAH44 48 533132 AC023120 701  1-113 1670-1968 2817-2876 3739-3958 4039-4861 8042-8236  9960-10253 10358-10487 11261-11363 12445-12554 17967-18436 19317-19470 19666-19900 20264-20363 20475-20779 25579-26082 26905-27377 27555-27983 28656-28997 29146-29769 30579-31170 31202-31625 32023-32698 34599-34740 34805-35520 35575-37925 HCFBJ91 55 531960 AC022075 702  1-164 479-585 1127-1211 3462-4318 5484-6362 HCFBJ91 55 531960 AC022075 703  1-157 HHFHP90 56 534615 AP001459 704   1-1102 HHFHP90 56 534615 AP000803 705   1-1101 HHFHP90 56 534615 AP001362 706   1-1102 HHFHP90 56 534615 AC073481 707  1-34 HHFHP90 56 534615 AC022488 708  1-102 1740-2313 2334-2673 3403-4504 4670-4849 5046-5195 5301-5477 5601-5841 6181-6671 7025-7084 HHFHP90 56 534615 AP001459 709  1-340 HHFHP90 56 534615 AP000803 710  1-340 HHFHP90 56 534615 AP001362 711  1-271 HHFHP90 56 534615 AC022488 712  1-549 HHFHP90 56 534615 AC022488 713  1-463 HHLAB07 58 532602 AC010764 714  1-750 HFOXE30 59 532120 AL031273 715  1-429 HFOXE30 59 532120 AC074234 716  1-397 HFOXE30 59 532120 AL031273 717  1-146 HFOXE30 59 532120 AL031273 718  1-175 HBJEL68 60 531924 AL159973 719  1-779 HBJEL68 60 531924 AL159973 720  1-217 236-688 HFOXA73 61 850699 AC005866 721  1-523 HFOXA73 61 850699 AC007618 722  1-522 HFIUR35 62 532062 AC063924 723  1-595 HFIUR35 62 532062 AC063924 724  1-375 HFPDE86 63 527728 AC007390 725  1-29 2108-2433 3020-3373 3513-3687 4189-5216 5348-5697 6109-9606  9821-10357 10998-11303 11348-11630 12256-12681 12718-13740 13752-14109 14197-14504 14635-15741 15884-16233 16283-16929 HFPDE86 63 527728 AC007390 726  1-429 HFPDE86 63 527728 AC007390 727  1-803

[0463] TABLE 1D cDNA Gene No. Clone ID Preferred Indication 1 HSIDU19 Digestive 2 HPRSB76 Reproductive 3 HTEIL66 Reproductive 4 HSNAY92 Cancer 5 HSABG21 Cancer 6 HSAXB32 Immune/Hematopoetic 7 HPEAD48 Reproductive 8 HPVAB94 Reproductive 9 HSAXB81 Immune/Hematopoetic 10 HSAYC21 Immune/Hematopoetic 11 HSLCU73 Musculoskeletal 12 HSSFZ70 Musculoskeletal 13 HTEIP36 Reproductive 14 HYBAY77 Immune/Hematopoetic, Musculoskeletal 15 HROAE78 Digestive 16 HSAVP17 Immune/Hematopoetic 17 HSIEA14 Digestive 18 HSNAQ47 Cancer 19 HODDN65 Reproductive 20 HPEAD79 Reproductive 21 HRDED19 Musculoskeletal 22 HSAYS89 Immune/Hematopoetic 23 HTODK73 Cancer 24 HSVAM10 Cancer 25 HSNBN57 Cancer 26 HSVBD22 Cancer 27 HSAWA27 Immune/Hematopoetic 28 HSFAH43 Cancer 29 HSPAA60 Digestive 30 HFAEF57 Neural/Sensory 31 HEGAH43 Digestive, Reproductive 32 HAGDG59 Cancer 33 HNGBX63 Immune/Hematopoetic 34 HE2AG50 Digestive, Mixed Fetal, Neural/Sensory 35 HCUIN80 Immune/Hematopoetic 36 HADCL29 Connective/Epithelial 37 HAPPS89 Cancer 38 HFGAH44 Cancer 39 HFIHZ96 Musculoskeletal 40 HFIUR10 Digestive, Immune/Hematopoetic, Musculoskeletal 41 HLDNA86 Cancer 42 HNGAN75 Immune/Hematopoetic 43 HCUIO20 Immune/Hematopoetic 44 HLTEF12 Cancer 45 HCFBJ91 Immune/Hematopoetic 46 HHFHP90 Cardiovascular 47 HLYCQ48 Immune/Hematopoetic 48 HHLAB07 Digestive, Immune/Hematopoetic 49 HFOXE30 Musculoskeletal 50 HBJEL68 Immune/Hematopoetic, Neural/Sensory 51 HFOXA73 Musculoskeletal 52 HFIUR35 Musculoskeletal 53 HFPDE86 Neural/Sensory

[0464] TABLE 1E cDNA Clone AA SEQ ID Gene No. ID NO: Y Biological Activity Exemplary Activity Assay Preferred Indication 2 HPRSB76 68 Production of IL-10 Assays for production of IL-10 Highly preferred indications and activation of T- and activation of T-cells are include allergy and asthma. cells. well known in the art and may Additional highly preferred be used or routinely modified indications include immune to assess the ability of and hematopoietic disorders polypeptides of the invention (e.g., as described below under (including antibodies and “Immune Activity”, and agonists or antagonists of the “Blood-Related Disorders”), invention) to stimulate or autoimmune diseases (e.g., inhibit production of IL-10 rheumatoid arthritis, systemic and/or activation of T-cells. lupus erythematosis, Crohn's. Exemplary assays that may be disease, multiple sclerosis used or routinely modified to and/or as described below), assess the ability of immunodeficiencies (e.g., as polypeptides and antibodies of described below), boosting a T the invention (including cell-mediated immune agonists or antagonists of the response, and suppressing a T invention) to modulate IL-10 cell-mediated immune production and/or T-cell response. proliferation include, for example, assays such as disclosed and/or cited in: Robinson, DS, et al., “Th-2 cytokines in allergic disease” Br Med Bull; 56 (4): 956-968 (2000), and Cohn, et al., “T- helper type 2 cell-directed therapy for asthma” Phramacology & Therapeutics; 88: 187-196 (2000); the contents of each of which are herein incorporated by reference in their entirety. Exemplary cells that may be used according to these assays include Th2 cells. IL10 secreted from Th2 cells may be measured as a marker of Th2 cell activation. Th2 cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that induce differentiation and activation of Th2 cells play a major role in the initiation and pathogenesis of allergy and asthma. Primary T helper 2 cells are generated in in vitro culture under Th2 polarizing conditions using peripheral blood lymphocytes isolated from cord blood. 5 HSABG21 71 Activation of Assays for the activation of Highly preferred indications 85: 6342-6346 (1988); Georas et al., Blood 92 (12): 4529-4538 (1998); Moffatt et al., Transplantation 69 (7): 1521-1523 (2000); Curiel et al., Eur J Immunol 27 (8): 1982-1987 (1997); and Masuda et al., J Biol Chem 275 (38): 29331-29337 (2000); the contents of each of which are herein incorporated by reference in the entirety. Exemplary cells that may be used according to these assays include mast cells. Mast cells are found in connective and mucosal tissues throughout the body, and their activation via immunoglobulin E-antigen, promoted by T-cell cytokines, is an important component of allergic and asthmatic disease. Furthermore, exemplary assays that may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to regulate signal transduction pathways through STAT-6 transcription factors in mast cells include assays disclosed and/or cited in: Sherman MA., “The role of STAT6 in mast cell IL-4 production” Immunol Rev; Feb; 179: 48-56 (2001); and, Masuda A, et al., “Interleukin- 15 induces rapid tyrosine phosphorylation of STAT6 and the expression of interleukin-4 in mouse mast cells” J Biol Chem; Sep 22; 275 (38): 29331-7 (2000); the contents of each of which are herein incorporated by reference in the entirety. 8 HPVAB94 74 Activation of Assays for the activation of Highly preferred indications transcription transcription through the include blood disorders (e.g., through NFAT Nuclear Factor of Activated T as described below under response element in cells (NFAT) response element “Immune Activity”, “Blood- immune cells (such are well-known in the art and Related Disorders”, and/or as T-cells). may be used or routinely ““Cardiovascular Disorders””). modified to assess the ability Highly preferred indications of polypeptides of the include autoimmune diseases invention (including antibodies (e.g., rheumatoid arthritis, and agonists or antagonists of systemic lupus erythematosis, the invention) to regulate multiple sclerosis and/or as NFAT transcription factors and described below), modulate expression of genes immunodeficiencies (e.g., as involved in described below), boosting a T immunomodulatory functions. cell-mediated immune Exemplary assays for response, and suppressing a T transcription through the cell-mediated immune NFAT response element that response. Additional highly may be used or routinely preferred indications include modified to test NFAT- inflammation and response element activity of inflammatory disorders. An polypeptides of the invention additional highly preferred (including antibodies and indication is infection (e.g., an agonists or antagonists of the infectious disease as described invention) include assays below under “Infectious disclosed in Berger et al., Gene Disease”). Preferred 66: 1-10 (1998); Cullen and indications include neoplastic Malm, Methods in Enzymol diseases (e.g., leukemia, 216: 362-368 (1992); Henthorn lymphoma, and/or as described et al., Proc Natl Acad Sci U.S.A. below under 85: 6342-6346 (1988); Serfling “Hyperproliferative et al., Biochim Biophys Acta Disorders”). Preferred 1498 (1): 1-18 (2000); De Boer indications include neoplasms et al., Int J Biochem Cell Biol and cancers, such as, for 31 (10): 1221-1236 (1999); example, leukemia, lymphoma, Fraser et al., Eur J Immunol and prostate, breast, lung, 29 (3): 838-844 (1999); and colon, pancreatic, esophageal, Yeseen et al., J Biol Chem stomach, brain, liver and 268 (19): 14285-14293 (1993), urinary cancer. Other preferred the contents of each of which indications include benign are herein incorporated by dysproliferative disorders and reference in its entirety. T pre-neoplastic conditions, such cells that may be used as, for example, hyperplasia, according to these assays are metaplasia, and/or dysplasia. publicly available (e.g., Preferred indications also through the ATCC). include anemia, pancytopenia, Exemplary human T cells that leukopenia, thrombocytopenia, may be used according to these Hodgkin's disease, acute assays include the SUPT cell lymphocytic anemia (ALL), line, which is a suspension plasmacytomas, multiple culture of IL-2 and IL-4 myeloma, Burkitt's lymphoma, responsive T cells. arthritis, AIDS, granulomatous disease, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, suppression of immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, asthma and allergy. 13 HTEIP36 79 Activation of Assays for the activation of Highly preferred indications transcription transcription through the include inflammation and through NFKB NFKB response element are inflammatory disorders. response element in well-known in the art and may Highly preferred indications immune cells (such be used or routinely modified include blood disorders (e.g., as T-cells). to assess the ability of as described below under polypeptides of the invention “Immune Activity”, “Blood- (including antibodies and Related Disorders”, and/or agonists or antagonists of the ““Cardiovascular Disorders””). invention) to regulate NFKB Highly preferred indications transcription factors and include autoimmune diseases modulate expression of (e.g., rheumatoid arthritis, immunomodulatory genes. systemic lupus erythematosis, Exemplary assays for multiple sclerosis and/or as transcription through the described below), and NFKB response element that immunodeficiencies (e.g., as may be used or routinely described below). An modified to test NFKB- additional highly preferred response element activity of indication is infection (e.g., polypeptides of the invention AIDS, and/or an infectious (including antibodies and disease as described below agonists or antagonists of the under “Infectious Disease”). invention) include assays Highly preferred indications disclosed in Berger et al., Gene include neoplastic diseases 66: 1-10 (1998); Cullen and (e.g., melanoma, leukemia, Malm, Methods in Enzymol lymphoma, and/or as described 216: 362-368 (1992); Henthorn below under et al., Proc Natl Acad Sci U.S.A. “Hyperproliferative 85: 6342-6346 (1988); Black et Disorders”). Highly preferred al., Virus Gnes 15 (2): 105-117 indications include neoplasms (1997); and Fraser et al., and cancers, such as, for 29 (3): 838-844 (1999), the example, melanoma, renal cell contents of each of which are carcinoma, leukemia, herein incorporated by lymphoma, and prostate, reference in its entirety. breast, lung, colon, pancreatic, Exemplary human T cells, esophageal, stomach, brain, such as the MOLT4, that may liver and urinary cancer. Other be used according to these preferred indications include assays are publicly available benign dysproliferative (e.g., through the ATCC). disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. Preferred indications also include anemia, pancytopenia, leukopenia, thrombocytopenia, Hodgkin's disease, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, AIDS, granulomatous disease, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, suppression of immune reactions to transplanted organs, asthma and allergy. 13 HTEIP36 79 Production of IL-10 Assays for production of IL-10 Highly preferred indications and activation of T- and activation of T-cells are include allergy and asthma. cells. well known in the art and may Additional highly preferred be used or routinely modified indications include immune to assess the ability of and hematopoietic disorders polypeptides of the invention (e.g., as described below under (including antibodies and “Immune Activity”, and agonists or antagonists of the “Blood-Related Disorders”), invention) to stimulate or autoimmune diseases (e.g., inhibit production of IL-10 rheumatoid arthritis, systemic and/or activation of T-cells. lupus erythematosis, Crohn's Exemplary assays that may be disease, multiple sclerosis used or routinely modified to and/or as described below), assess the ability of immunodeficiencies (e.g., as polypeptides and antibodies of described below), boosting a T the invention (including cell-mediated immune agonists or antagonists of the response, and suppressing a T invention) to modulate IL-10 cell-mediated immune production and/or T-cell response. proliferation include, for example, assays such as disclosed and/or cited in: Robinson, DS, et al., “Th-2 cytokines in allergic disease” Br Med Bull; 56 (4): 956-968 (2000), and Cohn, et al., “T- helper type 2 cell-directed therapy for asthma” Phramacology & Therapeutics; 88: 187-196 (2000); the contents of each of which are herein incorporated by reference in their entirety. Exemplary cells that may be used according to these assays include Th2 cells. IL10 secreted from Th2 cells may be measured as a marker of Th2 cell activation. Th2 cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that induce differentiation and activation of Th2 cells play a major role in the initiation and pathogenesis of allergy and asthma. Primary T helper 2 cells are generated in in vitro culture under Th2 polarizing conditions using peripheral blood lymphocytes isolated from cord blood. 19 HODDN65 85 Production of Assays for measuring Preferred embodiments of the ICAM-1 expression of ICAM-1 are invention include using well-known in the art and may polypeptides of the invention be used or routinely modified (or antibodies, agonists, or to assess the ability of antagonists thereof) in polypeptides of the invention detection, diagnosis, (including antibodies and prevention, and/or treatment of agonists or antagonists of the Inflammation, Vascular invention) to regulate ICAM-1 Disease, Athereosclerosis, expression. Exemplary assays Restenosis, and Stroke that may be used or routinely modified to measure ICAM-1 expression include assays disclosed in: Takacs P, et al, FASEB J, 15 (2): 279-281 (2001); and, Miyamoto K, et al., Am J Pathol, 156 (5): 1733-1739 (2000), the contents of each of which is herein incorporated by reference in its entirety. Cells that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include microvascular endothelial cells (MVEC). 20 HPEAD79 86 Regulation of Assays for the regulation of A highly preferred transcription via transcription through the indication is diabetes mellitus. DMEF1 response DMEF1 response element are An additional highly preferred element in well-known in the art and may indication is a complication adipocytes and pre- be used or routinely modified associated with diabetes (e.g., adipocytes to assess the ability of diabetic retinopathy, diabetic polypeptides of the invention nephropathy, kidney disease (including antibodies and (e.g., renal failure, agonists or antagonists of the nephropathy and/or other invention) to activate the diseases and disorders as DMEF1 response element in a described in the ““Renal reporter construct (such as that Disorders”” section below), containing the GLUT4 diabetic neuropathy, nerve promoter) and to regulate disease and nerve damage insulin production. The (e.g., due to diabetic DMEF1 response element is neuropathy), blood vessel present in the GLUT4 blockage, heart disease, stroke, promoter and binds to MEF2 impotence (e.g., due to diabetic transcription factor and another neuropathy or blood vessel transcription factor that is blockage), seizures, mental required for insulin regulation confusion, drowsiness, of Glut4 expression in skeletal nonketotic hyperglycemic- muscle. GLUT4 is the primary hyperosmolar coma, insulin-responsive glucose cardiovascular disease (e.g., transporter in fat and muscle heart disease, atherosclerosis, tissue. Exemplary assays that microvascular disease, may be used or routinely hypertension, stroke, and other modified to test for DMEF1 diseases and disorders as response element activity (in described in the adipocytes and pre-adipocytes) ““Cardiovascular Disorders”” by polypeptides of the section below), dyslipidemia, invention (including antibodies endocrine disorders (as and agonists or antagonists of described in the ““Endocrine the invention) include assays Disorders”” section below), disclosed in Thai, M.V., et al., J neuropathy, vision impairment Biol Chem, 273 (23): 14285-92 (e.g., diabetic retinopathy and (1998); Mora, S., et al., J Biol blindness), ulcers and impaired Chem, 275 (21): 16323-8 would healing, and infection (2000); Liu, M.L., et al., J Biol (e.g., infectious diseases and Chem, 269 (45): 28514-21 disorders as described in the (1994); ““Identification of a ““Infectious Diseases”” section 30-base pair regulatory below, especially of the element and novel DNA urinary tract and skin), carpal binding protein that regulates tunnel syndrome and the human GLUT4 promoter in Dupuytren's contracture). transgenic mice””, J Biol An additional highly preferred Chem. 2000 Aug indication is obesity and/or 4; 275 (31): 23666-73; Berger, et complications associated with al., Gene 66: 1-10 (1988); and, obesity. Additional highly Cullen, B., et al., Methods in preferred indications include Enzymol. 216: 362-368 (1992), weight loss or alternatively, the contents of each of which weight gain. Additional is herein incorporated by highly preferred indications are reference in its entirety. complications associated with Adipocytes and pre-adipocytes insulin resistance. that may be used according to these assays are publicly available (e.g., through the ATCC) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the mouse 3T3-L1 cell line which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of 3T3 fibroblasts developed through clonal isolation. These cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 23 HTODK73 89 Activation of Assays for the activation of Highly preferred indications transcription transcription through the include blood disorders (e.g., through NFAT Nuclear Factor of Activated T as described below under response in immune cells (NFAT) response element “Immune Activity”, “Blood- cells (such as T- are well-known in the art and Related Disorders”, and/or cells). may be used or routinely ““Cardiovascular Disorders””). modified to assess the ability Highly preferred indications of polypeptides of the include autoimmune diseases invention (including antibodies (e.g., rheumatoid arthritis, and agonists or antagonists of systemic lupus erythematosis, the invention) to regulate multiple sclerosis and/or as NFAT transcription factors and described below), modulate expression of genes immunodeficiencies (e.g., as involved in described below), boosting a T immunomodulatory functions. cell-mediated immune Exemplary assays for response, and suppressing a T transcription through the cell-mediated immune NFAT response element that response. Additional highly may be used or routinely preferred indications include modified to test NFAT- inflammation and response element activity of inflammatory disorders. An polypeptides of the invention additional highly preferred (including antibodies and indication is infection (e.g., an agonists or antagonists of the infectious disease as described invention) include assays below under “Infectious disclosed in Berger et al., Gene Disease”). Preferred 66: 1-10 (1998); Cullen and indications include neoplastic Malm, Methods in Enzymol diseases (e.g., leukemia, 216: 362-368 (1992); Henthorn lymphoma, and/or as described et al., Proc Natl Acad Sci U.S.A. below under 85: 6342-6346 (1988); Serfling “Hyperproliferative et al., Biochim Biophys Acta Disorders”). Preferred 1498 (1): 1-18 (2000); De Boer indications include neoplasms et al., Int J Biochem Cell Biol and cancers, such as, for 31 (10): 1221-1236 (1999); example, leukemia, lymphoma, Fraser et al., Eur J Immunol and prostate, breast, lung, 29 (3): 838-844 (1999); and colon, pancreatic, esophageal, Yeseen et al., J Biol Chem stomach, brain, liver and 268 (19): 14285-14293 (1993), urinary cancer. Other preferred the contents of each of which indications include benign are herein incorporated by dysproliferative disorders and reference in its entirety. T pre-neoplastic conditions, such cells that may be used as, for example, hyperplasia, according to these assays are metaplasia, and/or dysplasia. publicly available (e.g., Preferred indications also through the ATCC). include anemia, pancytopenia, Exemplary human T cells that leukopenia, thrombocytopenia, may be used according to these Hodgkin's disease, acute assays include the JURKAT lymphocytic anemia (ALL), cell line, which is a suspension plasmacytomas, multiple culture of leukemia cells that myeloma, Burkitt's lymphoma, produce IL-2 when stimulated. arthritis, AIDS, granulomatous disease, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, suppression of immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, asthma and allergy. 24 HSVAM10 90 Production of IFNgamma FMAT. IFNg plays A highly preferred IFNgamma using a a central role in the immune embodiment of the invention T cells system and is considered to be includes a method for a proinflammatory cytokine. stimulating the production of IFNg promotes TH1 and IFNg. An alternative highly inhibits TH2 differentiation; preferred embodiment of the promotes IgG2a and inhibits invention includes a method IgE secretion; induces for inhibiting the production of macrophage activation; and IFNg. Highly preferred increases MHC expression. indications include blood Assays for immunomodulatory disorders (e.g., as described proteins produced by T cells below under “Immune and NK cells that regulate a Activity”, “Blood-Related variety of inflammatory Disorders”, and/or activities and inhibit TH2 ““Cardiovascular Disorders””), helper cell functions are well and infection (e.g., viral known in the art and may be infections, tuberculosis, used or routinely modified to infections associated with assess the ability of chronic granulomatosus polypeptides of the invention disease and malignant (including antibodies and osteoporosis, and/or as agonists or antagonists of the described below under invention) to mediate “Infectious Disease”). Highly immunomodulation, regulate preferred indications include inflammatory activities, autoimmune disease (e.g., modulate TH2 helper cell rheumatoid arthritis, systemic function, and/or mediate lupus erythematosis, multiple humoral or cell-mediated sclerosis and/or as described immunity. Exemplary assays below), immunodeficiency that test for (e.g., as described below), immunomodulatory proteins boosting a T cell-mediated evaluate the production of immune response, and cytokines, such as Interferon suppressing a T cell-mediated gamma (IFNg), and the immune response. Additional activation of T cells. Such highly preferred indications assays that may be used or include inflammation and routinely modified to test inflammatory disorders. immunomodulatory activity of Additional preferred polypeptides of the invention indications include idiopathic (including antibodies and pulmonary fibrosis. Highly agonists or antagonists of the preferred indications include invention) include the assays neoplastic diseases (e.g., disclosed in Miraglia et al., J leukemia, lymphoma, Biomolecular Screening 4: 193-204 melanoma, and/or as described (1999); Rowland et al., below under ““Lymphocytes: a practical “Hyperproliferative approach”” Chapter 6: 138-160 Disorders”). Highly preferred (2000); Gonzalez et al., J Clin indications include neoplasms Lab Anal 8 (5): 225-233 (1995); and cancers, such as, for Billiau et al., Ann NY Acad example, leukemia, lymphoma, Sci 856: 22-32 (1998); Boehm melanoma, and prostate, et al., Annu Rev Immunol breast, lung, colon, pancreatic, 15: 749-795 (1997), and esophageal, stomach, brain, Rheumatology (Oxford) liver and urinary cancer. Other 38 (3): 214-20 (1999), the preferred indications include contents of each of which are benign dysproliferative herein incorporated by disorders and pre-neoplastic reference in its entirety. conditions, such as, for Human T cells that may be example, hyperplasia, used according to these assays metaplasia, and/or dysplasia. may be isolated using Preferred indications include techniques disclosed herein or anemia, pancytopenia, otherwise known in the art. leukopenia, thrombocytopenia, Human T cells are primary Hodgkin's disease, acute human lymphocytes that lymphocytic anemia (ALL), mature in the thymus and plasmacytomas, multiple express a T Cell receptor and myeloma, Burkitt's lymphoma, CD3, CD4, or CD8. These arthritis, AIDS, granulomatous cells mediate humoral or cell- disease, inflammatory bowel mediated immunity and may disease, sepsis, neutropenia, be preactivated to enhance neutrophilia, psoriasis, responsiveness to suppression of immune immunomodulatory factors. reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, asthma and allergy. 30 HFAEF57 96 Regulation of Assays for the regulation of A highly preferred transcription transcription through the FAS indication is diabetes mellitus. through the FAS promoter element are well- An additional highly preferred promoter element known in the art and may be indication is a complication in hepatocytes used or routinely modified to associated with diabetes (e.g., assess the ability of diabetic retinopathy, diabetic polypeptides of the invention nephropathy, kidney disease (including antibodies and (e.g., renal failure, agonists or antagonists of the nephropathy and/or other invention) to activate the FAS diseases and disorders as promoter element in a reporter described in the ““Renal construct and to regulate Disorders”” section below), transcription of FAS, a key diabetic neuropathy, nerve enzyme for lipogenesis. FAS disease and nerve damage promoter is regulated by many (e.g., due to diabetic transcription factors including neuropathy), blood vessel SREBP. Insulin increases FAS blockage, heart disease, stroke, gene transcription in livers of impotence (e.g., due to diabetic diabetic mice. This neuropathy or blood vessel stimulation of transcription is blockage), seizures, mental also somewhat glucose confusion, drowsiness, dependent. Exemplary assays nonketotic hyperglycemic- that may be used or routinely hyperosmolar coma, modified to test for FAS cardiovascular disease (e.g., promoter element activity (in heart disease, atherosclerosis, hepatocytes) by polypeptides microvascular disease, of the invention (including hypertension, stroke, and other antibodies and agonists or diseases and disorders as antagonists of the invention) described in the include assays disclosed in ““Cardiovascular Disorders”” Xiong, S., et al., Proc Natl section below), dyslipidemia, Acad Sci U.S.A., 97 (8): 3948-53 endocrine disorders (as (2000); Roder, K., et al., described in the ““Endocrine Eur J Biochem, 260 (3): 743-51 Disorders”” section below), (1999); Oskouian B, et al., neuropathy, vision impairment Biochem J, 317 (Pt 1): 257-65 (e.g., diabetic retinopathy and (1996); Berger, et al., Gene blindness), ulcers and impaired 66: 1-10 (1988); and, Cullen, wound healing, and infection B., et al., Methods in Enzymol. (e.g., infectious diseases and 216: 362-368 (1992), the disorders as described in the contents of each of which is ““Infectious Diseases”” section herein incorporated by below, especially of the reference in its entirety. urinary tract and skin), carpal Hepatocytes that may be used tunnel syndrome and according to these assays, such Dupuytren's contracture). as H4IIE cells, are publicly An additional highly preferred available (e.g., through the indication is obesity and/or ATCC) and/or may be complications associated with routinely generated. obesity. Additional highly Exemplary hepatocytes that preferred indications include may be used according to these weight loss or alternatively, assays include rat liver weight gain. Additional hepatoma cell line(s) inducible highly preferred indications are with glucocorticoids, insulin, complications associated with or cAMP derivatives. insulin resistance. 31 HEGAH43 97 Endothelial Cell Caspase Apoptosis. Assays for A highly preferred Apoptosis caspase apoptosis are well embodiment of the invention known in the art and may be includes a method for used or routinely modified to stimulating endothelial cell assess the ability of growth. An alternative highly polypeptides of the invention preferred embodiment of the (including antibodies and invention includes a method agonists or antagonists of the for inhibiting endothelial cell invention) to promote caspase growth. A highly preferred protease-mediated apoptosis. embodiment of the invention Induction of apoptosis in includes a method for endothelial cells supporting the stimulating endothelial cell vasculature of tumors is proliferation. An alternative associated with tumor highly preferred embodiment regression due to loss of tumor of the invention includes a blood supply. Exemplary method for inhibiting assays for caspase apoptosis endothelial cell proliferation. that may be used or routinely A highly preferred modified to test capase embodiment of the invention apoptosis activity of includes a method for polypeptides of the invention stimulating apoptosis of (including antibodies and endothelial cells. An agonists or antagonists of the alternative highly preferred invention) include the assays embodiment of the invention disclosed in Lee et al., FEBS includes a method for Lett 485 (2-3): 122-126 (2000); inhibiting (e.g., decreasing) Nor et al., J Vasc Res 37 (3): apoptosis of endothelial cells. 209-218 (2000); and Karsan A highly preferred and Harlan, J Atheroscler embodiment of the invention Thromb 3 (2): 75-80 (1996); includes a method for the contents of each of which stimulating angiogenisis. An are herein incorporated by alternative highly preferred reference in its entirety. embodiment of the invention Endothelial cells that may be includes a method for used according to these assays inhibiting angiogenesis. A are publicly available (e.g., highly preferred embodiment through commercial sources). of the invention includes a Exemplary endothelial cells method for reducing cardiac that may be used according to hypertrophy. An alternative these assays include bovine highly preferred embodiment aortic endothelial cells of the invention includes a (bAEC), which are an example method for inducing cardiac of endothelial cells which line hypertrophy. Highly blood vessels and are involved preferred indications include in functions that include, but neoplastic diseases (e.g., as are not limited to, described below under angiogenesis, vascular “Hyperproliferative permeability, vascular tone, Disorders”), and disorders of and immune cell extravasation. the cardiovascular system (e.g., heart disease, congestive heart failure, hypertension, aortic stenosis, cardiomyopathy, valvular regurgitation, left ventricular dysfunction, atherosclerosis and atherosclerotic vascular disease, diabetic nephropathy, intracardiac shunt, cardiac hypertrophy, myocardial infarction, chronic hemodynamic overload, and/or as described below under “Cardiovascular Disorders”). Highly preferred indications include cardiovascular, endothelial and/or angiogenic disorders (e.g., systemic disorders that affect vessels such as diabetes mellitus, as well as diseases of the vessels themselves, such as of the arteries, capillaries, veins and/or lymphatics). Highly preferred are indications that stimulate angiogenesis and/or cardiovascularization. Highly preferred are indications that inhibit angiogenesis and/or cardiovascularization. Highly preferred indications include antiangiogenic activity to treat solid tumors, leukemias, and Kaposi's sarcoma, and retinal disorders. Highly preferred indications include neoplasms and cancer, such as, Kaposi's sarcoma, hemangioma (capillary and cavernous), glomus tumors, telangiectasia, bacillary angiomatosis, hemangioendothelioma, angiosarcoma, haemangiopericytoma, lymphangioma, lymphangiosarcoma. Highly preferred indications also include cancers such as, prostate, breast, lung, colon, pancreatic, esophageal, stomach, brain, liver, and urinary cancer. Preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. Highly preferred indications also include arterial disease, such as, atherosclerosis, hypertension, coronary artery disease, inflammatory vasculitides, Reynaud's disease and Reynaud's phenomenom, aneurysms, restenosis; venous and lymphatic disorders such as thrombophlebitis, lymphangitis, and lymphedema; and other vascular disorders such as peripheral vascular disease, and cancer. Highly preferred indications also include trauma such as wounds, burns, and injured tissue (e.g., vascular injury such as, injury resulting from balloon angioplasty, and atheroschlerotic lesions), implant fixation, scarring, ischemia reperfusion injury, rheumatoid arthritis, cerebrovascular disease, renal diseases such as acute renal failure, and osteoporosis. Additional highly preferred indications include stroke, graft rejection, diabetic or other retinopathies, thrombotic and coagulative disorders, vascularitis, lymph angiogenesis, sexual disorders, age-related macular degeneration, and treatment/ prevention of endometriosis and related conditions. Additional highly preferred indications include fibromas, heart disease, cardiac arrest, heart valve disease, and vascular disease. Preferred indications include blood disorders (e.g., as described below under “Immune Activity”, “Blood- Related Disorders”, and/or ““Cardiovascular Disorders””). Preferred indications include autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosis, multiple sclerosis and/or as described below) and immunodeficiencies (e.g., as described below). Additional preferred indications include inflammation and inflammatory disorders (such as acute and chronic inflammatory diseases, e.g., inflammatory bowel disease and Crohn's disease), and pain management. 32 HAGDG59 98 Production of MIP1 MIP-1alpha FMAT. Assays A highly preferred alpha for immunomodulatory embodiment of the invention proteins produced by activated includes a method for dendritic cells that upregulate stimulating MIP1a production. monocyte/macrophage and T An alternative highly preferred cell chemotaxis are well embodiment of the invention known in the art and may be includes a method for used or routinely modified to inhibiting (e.g., reducing) assess the ability of MIP1a production. polypeptides of the invention A highly preferred (including antibodies and indication is infection (e.g., an agonists or antagonists of the infectious disease as described invention) to mediate below under “Infectious immunomodulation, modulate Disease”). chemotaxis, and modulate T Preferred indications cell differentiation. Exemplary include blood disorders (e.g., assays that test for as described below under immunomodulatory proteins “Immune Activity”, “Blood- evaluate the production of Related Disorders”, and/or chemokines, such as “Cardiovascular Disorders”). macrophage inflammatory Highly preferred indications protein 1 alpha (MIP-1a), and include autoimmune diseases the activation of (e.g., rheumatoid arthritis, monocytes/macrophages and T systemic lupus erythematosis, cells. Such assays that may be multiple sclerosis and/or as used or routinely modified to described below) and test immunomodulatory and immunodeficiencies (e.g., as chemotaxis activity of described below). Additional polypeptides of the invention highly preferred indications (including antibodies and include inflammation and agonists or antagonists of the inflammatory disorders. invention) include assays Preferred indications also disclosed in Miraglia et al., J include anemia, pancytopenia, Biomolecular Screening 4: 193-204 leukopenia, thrombocytopenia, (1999); Rowland et al., Hodgkin's disease, acute “Lymphocytes: a practical lymphocytic anemia (ALL), approach” Chapter 6: 138-160 plasmacytomas, multiple (2000); Satthaporn and myeloma, Burkitt's lymphoma Eremin, J R Coll Surg Ednb arthritis, AIDS, granulomatous 45 (1): 9-19 (2001); Drakes et al., disease, inflammatory bowel Transp Immunol 8 (1): 17-29 disease, sepsis, neutropenia, (2000); Verhasselt et al., J neutrophilia, psoriasis, Immunol 158: 2919-2925 suppression of immune (1997); and Nardelli et al., J reactions to transplanted Leukoc Biol 65: 822-828 organs and tissues, hemophilia (1999), the contents of each of hypercoagulation, diabetes which are herein incorporated mellitus, endocarditis, by reference in its entirety. meningitis, Lyme Disease, Human dendritic cells that may asthma, and allergy. be used according to these Preferred indications also assays may be isolated using include neoplastic diseases techniques disclosed herein or (e.g., leukemia, lymphoma, otherwise known in the art. and/or as described below Human dendritic cells are under “Hyperproliferative antigen presenting cells in Disorders”). Highly preferred suspension culture, which, indications include neoplasms when activated by antigen and cancers, such as, leukemia, and/or cytokines, initiate and lymphoma, prostate, breast, upregulate T cell proliferation lung, colon, pancreatic, and functional activities. esophageal, stomach, brain, liver, and urinary cancer. Other preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. 32 HAGDG59 98 T cell proliferation T Cell Proliferation. Assays A highly preferred for T cell proliferation, embodiment of the invention measured by tridiated (3H) includes a method for Thymidine incorporation, are stimulating T cell proliferation. well known in the art and may An alternative highly preferred be used or routinely modified embodiment of the invention to assess the ability of the includes a method for polypeptides of the invention inhibiting T cell proliferation. (including antibodies and Preferred indications include agonists or antagonists of the blood disorders (e.g., as invention) to mediate described below under immunomodulation, modulate “Immune Activity”, “Blood- T cell proliferation and Related Disorders”, and/or mediate humoral and/or cell- “Cardiovascular Disorders”), mediated immunity. and infection (e.g., an Exemplary assays that may be infectious disease as described used or routinely modified to below under “Infectious assess the ability of Disease”). Highly preferred polypeptides of the invention indications include (including antibodies and autoimmune diseases (e.g., agonists or antagonists of the rheumatoid arthritis, systemic invention) to modulate T cell lupus erythematosis, multiple proliferation, as measured by sclerosis and/or as described 3H Thymidine incorporation, below), immunodeficiencies include the assays disclosed in (e.g., as described below), Rowland et al., “Lymphocytes: boosting a T cell-mediated a practical approach” Chapter immune response, and 6: 138-160 (2000); Gansbacher suppressing a T cell-mediated and Zier, Cell Immunol immune response. Additional 117 (1): 22-34 (1988); and highly preferred indications Werfel et al., Allergy include inflammation and 52 (4): 465-469 (1997), the inflammatory disorders. contents of each of which are Highly preferred indications herein incorporated by include neoplastic diseases reference in its entirety. (e.g., leukemia, lymphoma, Human T cells that may be and/or as described below used according to these assays under “Hyperproliferative may be isolated using Disorders”). Highly preferred techniques disclosed herein or indications include neoplasms otherwise known in the art. and cancers, such as, for Human T cells are primary example, leukemia, lymphoma human lymphocytes that (e.g., T cell lymphoma, mature in the thymus and Burkitt's lymphoma, non- express a T cell receptor and Hodgkins lymphoma, CD3, CD4, or CD8. These Hodgkin's disease), melanoma, cells mediate humoral or cell- and prostate, breast, lung, mediated immunity and may colon, pancreatic, esophageal, be preactivated to enhance stomach, brain, liver and responsiveness to urinary cancer. Other preferred immunomodulatory factors. indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. Preferred indications include anemia, pancytopenia, leukopenia, thrombocytopenia, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, arthritis, AIDS, granulomatous disease, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, suppression of immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and asthma and allergy. 32 HAGDG59 98 T cell proliferation T Cell Proliferation. Assays A highly preferred for T cell proliferation, embodiment of the invention measured by tridiated (3H) includes a method for Thymidine incorporation, are stimulating T cell proliferation. well known in the art and may An alternative highly preferred be used or routinely modified embodiment of the invention to assess the ability of the includes a method for polypeptides of the invention inhibiting T cell proliferation. (including antibodies and Preferred indications include agonists or antagonists of the blood disorders (e.g., as invention) to mediate described below under immunomodulation, modulate “Immune Activity”, “Blood- T cell proliferation and Related Disorders”, and/or mediate humoral and/or cell- “Cardiovascular Disorders”), mediated immunity. and infection (e.g., an Exemplary assays that may be infectious disease as described used or routinely modified to below under “Infectious assess the ability of Disease”). Highly preferred polypeptides of the invention indications include (including antibodies and autoimmune diseases (e.g., agonists or antagonists of the rheumatoid arthritis, systemic invention) to modulate T cell lupus erythematosis, multiple proliferation, as measured by sclerosis and/or as described 3H Thymidine incorporation, below), immunodeficiencies include the assays disclosed in (e.g., as described below), Rowland et al., “Lymphocytes: boosting a T cell-mediated a practical approach” Chapter immune response, and 6: 138-160 (2000); Gansbacher suppressing a T cell-mediated and Zier, Cell Immunol immune response. Additional 117 (1): 22-34 (1988); and highly preferred indications Werfel et al., Allergy include inflammation and 52 (4): 465-469 (1997), the inflammatory disorders. contents of each of which are Highly preferred indications herein incorporated by include neoplastic diseases reference in its entirety. (e.g., leukemia, lymphoma, Human T cells that may be and/or as described below used according to these assays under “Hyperproliferative may be isolated using Disorders”). Highly preferred techniques disclosed herein or indications include neoplasms otherwise known in the art. and cancers, such as, for Human T cells are primary example, leukemia, lymphoma human lymphocytes that (e.g., T cell lymphoma, mature in the thymus and Burkitt's lymphoma, non- express a T cell receptor and Hodgkins lymphoma, CD3, CD4, or CD8. These Hodgkin's disease), melanoma, cells mediate humoral or cell- and prostate, breast, lung, mediated immunity and may colon, pancreatic, esophageal, be preactivated to enhance stomach, brain, liver and responsiveness to urinary cancer. Other preferred immunomodulatory factors. indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. Preferred indications include anemia, pancytopenia, leukopenia, thrombocytopenia, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, arthritis, AIDS, granulomatous disease, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, suppression of immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and asthma and allergy. 40 HFIUR10 106 Regulation of Assays for the regulation of A highly preferred viability and viability and proliferation of indication is diabetes mellitus. proliferation of cells in vitro are well-known in An additional highly preferred pancreatic beta the art and may be used or indication is a complication cells. routinely modified to assess associated with diabetes (e.g., the ability of polypeptides of diabetic retinopathy, diabetic the invention (including nephropathy, kidney disease antibodies and agonists or (e.g., renal failure, antagonists of the invention) to nephropathy and/or other regulate viability and diseases and disorders as proliferation of pancreatic beta described in the ““Renal cells. For example, the Cell Disorders”” section below), Titer-Glo luminescent cell diabetic neuropathy, nerve viability assay measures the disease and nerve damage number of viable cells in (e.g., due to diabetic culture based on quantitation neuropathy), blood vessel of the ATP present which blockage, heart disease, stroke, signals the presence of impotence (e.g., due to diabetic metabolically active cells. neuropathy or blood vessel Exemplary assays that may be blockage), seizures, mental used or routinely modified to confusion, drowsiness, test regulation of viability and nonketotic hyperglycemic- proliferation of pancreatic beta hyperosmolar coma, cells by polypeptides of the cardiovascular disease (e.g., invention (including antibodies heart disease, atherosclerosis, and agonists or antagonists of microvascular disease, the invention) include assays hypertension, stroke, and other disclosed in: Ohtani KI, et al., diseases and disorders as Endocrinology, 139 (1): 172-8 described in the (1998); Krautheim A, et al, ““Cardiovascular Disorders”” Exp Clin Endocrinol Diabetes, section below), dyslipidemia, 107 (1): 29-34 (1999), the endocrine disorders (as contents of each of which is described in the ““Endocrine herein incorporated by Disorders”” section below), reference in its entirety. neuropathy, vision impairment Pancreatic cells that may be (e.g., diabetic retinopathy and used according to these assays blindness), ulcers and impaired are publicly available (e.g., wound healing, and infection through the ATCC) and/or (e.g., infectious diseases and may be routinely generated. disorders as described in the Exemplary pancreatic cells that ““Infectious Diseases”” section may be used according to these below, especially of the assays include HITT15 Cells. urinary tract and skin), carpal HITT15 are an adherent tunnel syndrome and epithelial cell line established Dupuytren's contracture). from Syrian hamster islet cells An additional highly preferred transformed with SV40. These indication is obesity and/or cells express glucagon, complications associated with somatostatin, and obesity. Additional highly glucocorticoid receptors. The preferred indications include cells secrete insulin, which is weight loss or alternatively, stimulated by glucose and weight gain. glucagon and suppressed by Additional highly preferred somatostatin or indications are complications glucocorticoids. ATTC# CRL- associated with insulin 1777 Refs: Lord and resistance. Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. U.S.A 78: 4339-4343, 1981. 41 HLDNA86 107 Activation of Kinase assay. Kinase assays, A highly preferred Adipocyte ERK for example an Elk-1 kinase embodiment of the invention Signaling Pathway assay, for ERK signal includes a method for transduction that regulate cell stimulating adipocyte proliferation or differentiation proliferation. An alternative are well known in the art and highly preferred embodiment may be used or routinely of the invention includes a modified to assess the ability method for inhibiting of polypeptides of the adipocyte proliferation. A invention (including antibodies highly preferred embodiment and agonists or antagonists of of the invention includes a the invention) to promote or method for stimulating inhibit cell proliferation, adipocyte differentiation. An activation, and differentiation. alternative highly preferred Exemplary assays for ERK embodiment of the invention kinase activity that may be includes a method for used or routinely modified to inhibiting adipocyte test ERK kinase-induced differentiation. A highly activity of polypeptides of the preferred embodiment of the invention (including antibodies invention includes a method and agonists or antagonists of for stimulating (e.g., the invention) include the increasing) adipocyte assays disclosed in Forrer et al., activation. An alternative Biol Chem 379 (8-9): 1101-1110 highly preferred embodiment (1998); Le Marchand- of the invention includes a Brustel Y, Exp Clin method for inhibiting the Endocrinol Diabetes activation of (e.g., decreasing) 107 (2): 126-132 (1999); and/or inactivating adipocytes. Kyriakis JM, Biochem Soc Highly preferred indications Symp 64: 29-48 (1999); Chang include endocrine disorders and Karin, Nature (e.g., as described below under 410 (6824): 37-40 (2001); and ““Endocrine Disorders””). Cobb MH, Prog Biophys Mol Highly preferred indications Biol 71 (3-4): 479-500 (1999); also include neoplastic the contents of each of which diseases (e.g., lipomas, are herein incorporated by liposarcomas, and/or as reference in its entirety. described below under Mouse adipocyte cells that “Hyperproliferative may be used according to these Disorders”). Preferred assays are publicly available indications include blood (e.g., through the ATCC). disorders (e.g., hypertension, Exemplary mouse adipocyte congestive heart failure, blood cells that may be used vessel blockage, heart disease, according to these assays stroke, impotence and/or as include 3T3-L1 cells. 3T3-L1 described below under is an adherent mouse “Immune Activity”, preadipocyte cell line that is a “Cardiovascular Disorders”, continuous substrain of 3T3 and/or “Blood-Related fibroblast cells developed Disorders”), immune disorders through clonal isolation and (e.g., as described below under undergo a pre-adipocyte to ““Immune Activity””), neural adipose-like conversion under disorders (e.g., as described appropriate differentiation below under ““Neural Activity conditions known in the art. and Neurological Diseases””), and infection (e.g., as described below under “Infectious Disease”). A highly preferred indication is diabetes mellitus. An additional highly preferred indication is a complication associated with diabetes (e.g., diabetic retinopathy, diabetic nephropathy, kidney disease (e.g., renal failure, nephropathy and/or other diseases and disorders as described in the ““Renal Disorders”” section below), diabetic neuropathy, nerve disease and nerve damage (e.g., due to diabetic neuropathy), blood vessel blockage, heart disease, stroke, impotence (e.g., due to diabetic neuropathy or blood vessel blockage), seizures, mental confusion, drowsiness, nonketotic hyperglycemic- hyperosmolar coma, cardiovascular disease (e.g., heart disease, atherosclerosis, microvascular disease, hypertension, stroke, and other diseases and disorders as described in the ““Cardiovascular Disorders”” section below), dyslipidemia, endocrine disorders (as described in the ““Endocrine Disorders”” section below), neuropathy, vision impairment (e.g., diabetic retinopathy and blindness), ulcers and impaired wound healing, infection (e.g., infectious diseases and disorders as described in the ““Infectious Diseases”” section below (particularly of the urinary tract and skin). An additional highly preferred indication is obesity and/or complications associated with obesity. Additional highly preferred indications include weight loss or alternatively, weight gain. Additional highly preferred indications are complications associated with insulin resistance. Additional highly preferred indications are disorders of the musculoskeletal systems including myopathies, muscular dystrophy, and/or as described herein. Additional highly preferred indications include, hypertension, coronary artery disease, dyslipidemia, gallstones, osteoarthritis, degenerative arthritis, eating disorders, fibrosis, cachexia, and kidney diseases or disorders. Preferred indications include neoplasms and cancer, such as, lymphoma, leukemia and breast, colon, and kidney cancer. Additional preferred indications include melanoma, prostate, lung, pancreatic, esophageal, stomach, brain, liver, and urinary cancer. Highly preferred indications include lipomas and liposarcomas. Other preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. 51 HFOXA73 117 Production of IL-10 Assays for production of IL-10 Highly preferred indications and activation of T- and activation of T-cells are include allergy and asthma. cells. well known in the art and may Additional highly preferred be used or routinely modified indications include immune to assess the ability of and hematopoietic disorders polypeptides of the invention (e.g., as described below under (including antibodies and “Immune Activity”, and agonists or antagonists of the “Blood-Related Disorders”), invention) to stimulate or autoimmune diseases (e.g., inhibit production of IL-10 rheumatoid arthritis, systemic and/or activation of T-cells. lupus erythematosis, Crohn's Exemplary assays that may be disease, multiple sclerosis used or routinely modified to and/or as described below), assess the ability of immunodeficiencies (e.g., as polypeptides and antibodies of described below), boosting a T the invention (including cell-mediated immune agonists or antagonists of the response, and suppressing a T invention) to modulate IL-10 cell-mediated immune production and/or T-cell response. proliferation include, for example, assays such as disclosed and/or cited in: Robinson, DS, et al., “Th-2 cytokines in allergic disease” Br Med Bull; 56 (4): 956-968 (2000), and Cohn, et al., “T- helper type 2 cell-directed therapy for asthma” Phramacology & Therapeutics; 88: 187-196 (2000); the contents of each of which are herein incorporated by reference in their entirety. Exemplary cells that may be used according to these assays include Th2 cells. IL10 secreted from Th2 cells may be measured as a marker of Th2 cell activation. Th2 cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that induce differentiation and activation of Th2 cells play a major role in the initiation and pathogenesis of allergy and asthma. Primary T helper 2 cells are generated in in vitro culture under Th2 polarizing conditions using peripheral blood lymphocytes isolated from cord blood.

[0465] TABLE 2 SEQ Score/ cDNA Contig ID Analysis PFam/NR Accession Percent Clone ID ID: NO: X Method PFam/NR Description Number Identity NT From NT To HSIDU19 520372 11 blastx.2 NA/PO4 sp|Q9Y2C5|Q9Y2C5 100%  9 461 COTRANSPORTER HOMOLOG. HPRSB76 526310 12 blastx.2 POTENTIAL sp|O54827|AT5A_MOUSE 83% 112 570 PHOSPHOLIPID- 40% 2 364 TRANSPORTING ATPASE VA (EC 1 HTEIL66 520125 13 WUblastx.64 (AK015053) putative dbj|BAB29693.1| 51% 36 509 [Mus musculus] HSNAY92 526730 14 blastx.2 ubiquitous TPR motif pir|T02214|T02214 100%  18 347 isoform Y - human HSABG21 526366 15 WUblastx.64 (AK021539) unnamed dbj|BAB13840.1| 97% 413 556 protein product [Homo 99% 48 416 sapiens] HSAXB32 520470 16 blastx.2 CDNA FLJ20378 FIS, sp|Q9NX85|Q9NX85 72% 1029 751 CLONE KAIA0536. 52% 754 692 HSAXB81 520471 19 blastx.2 CDNA FLJ20489 FIS, sp|Q9NX17|Q9NX17 54% 772 458 CLONE KAT08285. HSLCU73 520237 21 blastx.2 PRO1722. sp|Q9P195|Q9P195 62% 767 600 56% 587 468 50% 622 491 76% 478 416 51% 414 322 HYBAY77 520394 24 blastx.2 SIMILAR TO POGO sp|Q13537|Q13537 47% 447 1 ELEMENT. HSAVP17 519846 26 blastx.2 CDNA: FLJ21463 fis, sp|BAB15071|BAB15071 69% 691 509 clone COL04765. 73% 828 694 HSIEA14 520387 27 blastx.2 UNNAMED PORTEIN sp|Q9N083|Q9N083 66% 667 479 PRODUCT. 58% 779 672 HSNAQ47 847453 28 WUblastx.64 predicted using emb|CAB04264.1| 35% 95 784 Genefinder˜cDNA EST yk38g9.3 comes from this 11111111 this gene [Caenorhabditis elegans] HSNAQ47 526779 64 blastx.2 hypothetical protein pir|T21713|T21713 35% 99 788 F33H2.2 - Caenorhabditis elegans HODDN65 520348 29 blastx.2 UNNAMED PORTEIN sp|Q9N083|Q9N083 67% 660 493 PRODUCT. 74% 743 663 HPEAD79 520202 30 blastx.2 PRO2550. sp|AAG35515|AAG35515 49% 808 497 HTODK73 526021 33 blastx.2 CDNA FLJ13348 fis, sp|BAB14566|BAB14566 80% 21 401 clone OVARC1002127, 100%  567 707 weakly similar to 1 43% 4 189 61% 418 519 93% 404 448 71% 433 474 HSAWA27 520302 37 blastx.2 PRO2550. sp|AAG35515|AAG35515 78% 531 418 55% 649 521 60% 422 336 66% 396 352 HSFAH43 520399 38 blastx.2 HYPOTHETICAL sp|Q9Y5K0|Q9Y5K0 81% 9 239 149.0 KDA PROTEIN. 89% 241 351 HFAEF57 534142 40 blastx.2 Hypothetical 15.8 kDa sp|AAG17270|AAG17270 47% 601 425 protein. HEGAH43 532596 41 blastx.2 (AL360078) bA530N10.1 emb|CAC17684.1| 100%  29 361 (novel protein) [Homo sapiens] HAGDG59 534165 42 HMMER PFAM: short chain PF00106 182.2 232 795 2.1.1 dehydrogenase HAGDG59 534165 42 WUblastx.64 (AF126780) retinal short- gb|AAF06939.1|AF126780_1 96% 124 1023 chain dehydrogenase/reductase retSDR2 [Homo sapiens] HCUIN80 534128 45 blastx.2 CDNA: FLJ21463 fis, sp|BAB15071|BAB15071 78% 825 562 clone COL04765. HAPPS89 532135 47 WUblastx.64 (AF010144) neuronal gb|AAC08737.1| 64% 819 658 thread protein AD7c-NTP 64% 809 657 [Homo sapiens] 55% 600 523 45% 667 602 68% 668 603 70% 810 541 HFIUR10 532060 50 blastx.2 CDNA: FLJ21463 fis, sp|BAB15071|BAB15071 54% 497 339 clone COL04765. 90% 523 494 HLDNA86 535730 51 blastx.2 (BC000828) Unknown gb|AAH00828.1|AAH00828 81% 45 218 (protein for IMAGE: 3455200) [Homo sapiens] HLTEF12 532354 54 blastx.2 isocitrate dehydrogenase pir|S57499|S57499 98% 469 1188 (NADP+) (EC 1.1.1.42) 98% 206 475 precursor, mitochondrial - human HCFBJ91 531960 55 blastx.2 CDNA: FLJ21394 fis, sp|BAB15056|BAB15056 68% 764 615 clone COL03536. 63% 877 758 40% 654 520 47% 761 705 HHFHP90 534615 56 blastx.2 PRO2550. sp|AAG35515|AAG35515 74% 982 800 79% 1101 970 HLYCQ48 527757 57 blastx.2 CDNA FLJ20489 FIS, sp|Q9NX17|Q9NX17 62% 755 471 CLONE KAT08285. HBJEL68 531924 60 blastx.2 PRO0117 PROTEIN. sp|Q9UI85|Q9UI85 59% 774 634 72% 643 611 HFPDE86 527728 63 blastx.2 PRO2822. sp|Q9P147|Q9P147 72% 737 516

[0466] TABLE 3 SEQ cDNA ID Contig EST Disclaimer Clone ID NO: X ID: Range of a Range of b Accession #’s HSIDU19 11 520372 1-666 15-680 AI056789, AK024903, AB020527, AL391194, AL391194, and AL391194. HPRSB76 12 526310 1-727 15-741 AA447438, T09355, AB011138, and Y09954. HTEIL66 13 520125 1-605 15-619 AC067727. HSNAY92 14 526730 1-597 15-611 R64076, AA730371, R27397, AU139244, H71069, BF089099, AC010877, AC005820, AF000994, AF000992, AF000993, AJ002730, AF132076, AC002538, AL031427, AL031294, AL031686, AC006038, and AL009177. HSABG21 15 526366 1-571 15-585 AA421255, AU145857, AA374532, AK021539, AK023067, and AK021849. HSAXB32 16 520470 1-1026 15-1040 AA635135, BF803143, BE273825, AW162314, AW410844, AW265468, AW020150, AL036896, AW275432, BG180320, AI732690, AL121039, AI702049, AA526542, AV732057, AW504667, AA601376, AI065031, AW162332, AU151751, AI251024, AI926102, AW975150, AV760560, AW963489, AW974923, N72509, BE677164, AI521525, AW021674, AA640305, AA595661, AL042667, AL042670, AA525753, AI590442, AI890297, AI924950, AA218684, AI754926, AA171400, AV758849, BE244308, AI078409, AW338376, AA577706, AI744963, AV683406, AW277000, AA425283, AW277093, AA557945, AW157424, AA533066, AA533660, AI254267, AW813775, BF880881, AW970588, AW975239, AA084320, AU147226, M77888, BE138520, BG059139, AV730440, AA324108, AL044701, AA493546, AW169183, AI732327, AW151247, AA179364, BE328286, AI174703, AW020094, AI962030, BE967607, BE140949, AI064968, BG029528, AV761486, AW272389, AV712092, AV760508, AA313025, BF589864, BE676765, BF575954, AW265006, AI801563, AI049999, AI547110, AW855527, AW963552, AA935827, AI791659, AI872229, AI653776, AV755245, AI567676, BE154381, AI242236, AI955447, AI031759, AV717475, AW518140, AV710695, BG152746, AA601674, AW082104, BG236484, AV729908, AW020612, BE049409, BF530611, AA847341, AI538404, AI745666, AA573067, AI613459, BG059314, BE139230, AW328331, N99245, AW327852, AW192930, AI090377, AA610381, AI434103, BE139673, BG223498, AV743869, AW576299, BF846619, AI281622, AA112864, AW272815, AI521019, AL138262, AW177869, AI473671, AV758870, AV757180, AI445699, BF725844, AI500563, BF834298, AU155104, BF668217, AW675677, AW409626, AL041375, BF243651, AI884404, BG250794, AI537368, AC016655, AC010419, AP000694, AP001725, AL109921, AP000359, AL031311, AL021707, L35532, AL136139, AL117381, AC002115, AC002369, AC010422, AC002470, AL034420, AC022432, AL109798, AC006483, AL136131, AL034422, AP000553, AL133215, AL353748, AC008892, AL132765, AL157817, Z93244, AC006994, AL031658, AC004242, AL365444, AP000239, Z86090, AL136304, AL022316, AC005540, AC004771, AP000095, AJ400877, AP001670, AP001705, AC004816, AL049776, AC005808, AL117348, AL157768, AL008630, AC003665, AL353680, Z98941, AL121914, U52111, AL121983, AL159970, AC008747, AC005091, AC005049, AC002553, AC004206, AC005940, AC003007, AB014080, AC008969, AL157877, AC008083, AL360227, AC007324, AC006511, AL353804, AB041992, AL009181, AC025593, AC007845, AC004884, AC005098, AC005179, AB023048, AL356801, AL050318, Z97055, AP001630, AF030876, AL049694, AC003969, AC008897, Z97056, AL158040, AC009086, AL050335, AL356215, AL109984, AP000514, AL135927, AC007227, AL117329, AL117258, AF168787, AC016026, AL133445, Z99716, Z93017, AL356379, AC003037, AC022402, AP002529, U47924, AC006974, AC008569, AC009244, AC005304, AC010789, AL136418, AC004890, AC002565, AP001717, AC008687, AC007688, AJ277557, AC005180, Z83844, AC006071, AL137818, AL035693, AC005726, AC006162, AP001748, AC004913, AL035072, AL132795, AP000501, AL132653, AC005841, AC005519, Z70289, AC011480, AL359457, AC006536, AC005838, AL109806, AL137783, AP000555, AC005274, AC005776, AL008582, AL353807, AP000511, AL031427, AC005736, AC008543, AL049712, AC002351, AC000115, AL133404, AL357374, AC005529, AC026776, AP000031, AL080243, AP000030, AC005937, AP001711, Z97632, AC005261, AP000252, AF205588, AL139824, AL122020, AC007055, AC008267, AP000211, AP000133, Z83840, AC006079, AL031846, AP000113, AC002073, AC007686, M89651, AL354720, AC010526, AC009134, AC011479, AC004826, AC011490, AF196972, AL136969, AC008068, AL022721, AL050349, AL096677, AC005527, AC008745, U91325, AC020916, AC007934, AC005484, AL121712, AC067969, AC024561, AC011495, AP000552, AC008784, AC005793, AC010326, AC011444, AL008732, AC004150, AF111168, AL138713, AF031075, AL035659, AC009314, AC010553, AL031347, AC005324, AC008536, AC000025, AL109797, AF141309, AC011442, AL358777, AC007225, AC008403, AC007272, AC007386, AC006060, AL157372, AL138759, AC004997, AC010271, AL135905, AC008753, AF015720, AP000503, AF134726, AC005829, AL031729, AC005913, Z95116, AL138807, AC002991, AF217403, AF196779, AC005531, AL137139, AC005632, AC002395, AL022336, AP001973, AC027536, AC011957, AC010419, AC010419, AC010419, AC016655, AC016655, AC016655, AC002369, AC002369, AC018548, AC018548, and AC018548. HPEAD48 17 520367 1-611 15-625 AW964468, D80045, AW966389, AW949645, AW966330, AW949642, AW964532, AW965158, AW949643, AV738340, AV744012, AW975618, AV744690, AW366296, AV702035, AV723097, C14389, AV724520, AV742048, AW962395, D51799, AW964488, AV719468, D59502, AW375405, D80195, AV720791, AA305578, AW966050, AW965185, AW965197, AW959597, AV742732, AV718800, D80164, AV742050, AW966053, AW966013, AW949658, AW975621, AW966054, AV719783, AW966534, AV720464, AI905856, AW960465, AW949654, AV705134, AW966022, AW966075, AW966041, C15076, AW966065, AW973541, D80038, AW978648, AW959062, AV719324, AV718440, AV720028, AW975613, D59467, AW965196, AW965184, AW965175, D59275, AV718770, AW966030, AV719188, D80227, AW964477, AW966062, AW949641, AW949646, AW959570, AW966029, AV718707, AV705869, AV699927, AW973334, AW966531, AW978634, AW956434, D58283, D80022, AW965163, D80166, AW959799, AW966059, D80193, AW960473, D59859, D59619, D80210, D80391, AV704180, AW960553, D80240, C14331, AW973474, AV719822, AV718692, D59787, D51423, AW973488, AW978661, AV720211, AV718844, D81030, D80253, AV718489, AV720203, AW964756, AW973307, D80043, AV723927, AW177440, AV718938, AW949656, AV718633, AW959628, AW965177, AW975605, AW973485, AV718931, AV720878, AV719557, AV720731, AW973482, AV699447, AW958992, AW958993, AV722801, AW959136, AV699550, AW962082, D80269, AW959202, AV720812, D80212, D80196, AW949657, D80188, D80024, D50979, D80219, AV706147, AW753067, D80268, AW966043, AW949655, AW959582, D59927, D59610, AV720533, D57483, AA305409, AV719628, D80378, D80366, AV741220, D51022, AV706229, AW973330, AW962245, AV721386, AW973447, D59889, D50995, AA514188, D81026, AV727978, AW960454, AW949653, AW949631, AW949618, AW959469, AW964737, AW966032, AV720150, AW960532, AW956397, AW949629, D51060, AW949633, AW949632, AV700889, AV699866, AV701123, C14429, AW973490, AW752082, AV726812, AW753053, AW965176, AW966023, D80241, AW177501, AW960564, AW177511, AA514186, AW178893, AW949630, AW960504, AV699746, AW375406, AV700229, AV699682, AW973465, AW960474, D80248, AV718530, AV703542, AV701004, D80522, AW975623, T03269, AV720654, AV699669, AW960570, AW360811, AW179328, AW964541, AW973473, AW973445, AV720220, C75259, AV720616, AW966332, D80251, AV719000, C14014, AW352117, D80133, AW960514, AW966399, AW966333, AW966331, AW753041, AV718908, D58253, AV742001, AV742667, AV742022, AL136136, A62298, A62300, AX047063, AX047064, AR070327, A82595, AX047062, A84916, AX020191, AX035434, AR018138, AX020190, Y17188, AR016808, AR074545, AX027925, AJ132110, AX033851, AR092424, A94995, AF058696, AB028859, AR008278, AX021518, AR087649, Y17187, X67155, D26022, A25909, AX028130, A67220, D89785, A78862, D34614, AJ302649, Y12724, AR016514, D88547, AR077702, A43190, AR060385, X82626, AB002449, AR025207, AR008443, AR038669, I50126, I50132, I50128, AR091537, I50133, A30438, AJ294956, AR066488, AF260572, AR060138, A45456, A44171, A26615, AR052274, AX015396, AB012117, I19525, AR074139, Y09669, A43192, AR066490, I14842, AR066487, AR074136, I18367, X68127, AR054175, AX014811, AJ287395, A85396, AR074141, AR066482, D50010, AR088705, AX042372, A85477, D88507, A86792, A63261, X93549, AR008277, AR008281, A70867, AR008408, AR062872, AR093385, I79511, AR016691, AR016690, U46128, A64136, A68321, D13509, AR060133, AR050070, AF135125, AJ000347, I32384, U79457, AF123263, AR032065, and AR008382. HPVAB94 18 526749 1-805 15-819 BF370024, AW449056, AF131217, AL163247, AF165147, AF131217, and AF165147. HSAXB81 19 520471 1-768 15-782 BF367762, AA904211, D29019, AA469451, BF804385, AL079734, AL353771, AL133245, AL031659, AC005520, AL121653, Z49236, AF196779, AL031681, AJ295844, AC008379, AP000513, AL135744, AP001726, AC015651, AL079342, AC004019, AP000694, AL034372, AC010553, AL109804, AP000359, AC007688, AL021407, AC004824, AF254983, Z84469, AC004983, AC002418, AL109984, AC011469, AC011742, AC004386, AC008753, AC005089, AC007773, AF168787, Z85987, AL132639, AC006483, AL353596, AL049829, AF196969, AC004898, AC027319, AL163201, AL136170, AC007845, AL035458, AL138717, AP001748, AC004491, AL033529, AC007404, AC009958, AL445472, AC021016, AC007731, AL031282, L48038, AC006480, AC005261, Z82202, AC009194, AL021391, AC011464, AC005500, AL049776, AC004106, Z82190, AC008649, AL109936, AL022476, AC005954, AC011489, AC007383, AL023583, AL109743, AC004975, AC009086, AP001760, AC009408, Z82214, L78833, AC011248, AC002115, AC000052, AL035681, AL109797, Z84466, AC008745, AC008760, U82671, AC007298, AL139343, AX039602, AP000095, AL022163, AC004882, AC009298, AC005512, AC019213, AC019213, AC019213, AL353771, AL353771, AL353771, AL354721, and AL354721. HSAYC21 20 526188 1-641 15-655 BE856451, AA653742, AC008372, AC000353, AC011097, AC011097, AC011097, and AC019279. HSLCU73 21 520237 1-784 15-798 AI866487, AV759172, AW965008, AA704393, AW440545, AL119691, AI963720, AL135377, AI282479, AA587604, BG109996, AI745151, AV762957, AI732151, AW103758, AW088846, AV710066, AV735495, AW028429, AA167055, BF592311, AV742057, BF826830, AW963463, AV728425, AI270117, AW504104, AV734149, X87344, AC006512, AC022392, AL445490, AL159168, AL355094, AC002553, AC006328, AC019187, AC012064, AL353706, AC006011, AC015651, AC007690, U73634, AC005630, AC010724, AC005899, AC010725, AP001714, U50871, AC004815, AL109921, AL445217, AC009399, AL133453, AL162390, AP000555, AP001717, AC006482, AP000048, AF165926, U85195, AL049776, AC009403, AC005098, Z81369, AP000553, AE000658, U95742, AL079295, AL133353, AC004166, U96629, AP001711, AC005512, AC003983, AL109798, AC006449, AL135927, AC007227, AC004996, AC003029, AF129756, AC004824, AC005844, AC020629, AL136526, AC022078, AL136418, AC007386, AF217403, AL109758, Z84469, AC011742, AC005971, AC011475, AL050308, AC005907, AC004858, AP000037, AP000105, AC004890, AC011497, AL136979, AP000557, AC005089, AC009307, AC007216, AC008379, AC005257, AL109935, AL022322, AC008115, AL033529, Z84488, AC000025, AC005784, AL079335, AC011484, AC007842, AC004019, AC023511, AL022328, AC009086, AF219991, AF196779, AC009228, AC005104, AL022320, AP000114, AP000046, AL009183, AC006285, AP001710, Z94044, AC002996, AC005231, AL031588, AC022311, AL132639, AC006013, AL035587, AC025593, AC008569, AF053356, AC011811, AC009244, AC005020, AX039602, AC006515, AC011445, AC000026, Z93015, AC010267, U91321, AL136365, Z86090, AB017654, AC007546, AC006211, AC006001, AC027319, AL022327, AC007536, AC011464, AC004971, AL133232, AC018639, Z93930, AC004526, AF131216, AL138759, AL031733, AC004644, Z82208, AL133174, AC009530, AC005280, AL137818, AC004686, AP001053, AP001670, AC007242, AL160175, AL021391, AL135749, AC005722, AL035249, U47924, AC007055, AL118520, AL161670, AF207550, AL137119, AC020906, AC005696, AC006950, U91323, AL096701, U95739, AC006275, AC005924, AC004257, AL121655, AL049795, AL035684, AC008745, AC011495, AL109804, AC004967, AP000509, AF064861, AF109907, AC006115, AL080243, AC000052, AP001695, AC005632, AC006511, AF134726, AC004802, AF196972, AL049759, AL158830, AF196969, AC016027, AC011527, AC006121, AL163279, AL031681, AL121893, U80017, U95090, AL022343, AL109914, U62293, AC016830, AC002470, AC005081, AL022316, AC002544, AC005261, AC011470, AL021393, AL359238, AC005988, AC006538, AC009475, AC002059, AL109743, AL034380, AC004253, Z93017, AP000961, AC010422, AL049589, AL031281, AC008764, AP001688, Z93020, AL135839, AC009311, AC006077, U07561, AC005056, AL136137, AC010311, AL138784, and AL138784. HSSFZ70 22 526499 1-632 15-646 AI207437, BE877529, AI567204, AI478230, Z98486, BE908499, AW827226, AV705400, BE880328, BE873944, AI005023, AI953563, BF724930, BF032694, AA654003, BE875705, AA292238, AV702832, AV704592, AV702861, BE873725, AW410260, AW410829, AI560202, BE907715, BE881031, BG176797, AA806061, AA166890, AI564171, AL037085, BG177029, AW088118, AW411537, AI625499, BE878269, AI919534, AL037693, AA088891, BE877210, AA225922, BE874251, AA575974, AI963858, AI609179, AI634594, AA180745, AW601571, BE880300, AI699885, AA766508, BG165872, AW103371, AV722918, H29578, AI289160, AA018554, AI888869, AI579931, BE257077, AI799374, AW162189, AI625489, AW409568, AA088755, AA565315, AV710589, BE875112, BF672427, AI743700, AI699797, BF983585, AA283870, AI650301, AA019432, AW411050, BE908424, BG179111, AI913315, AA844225, AI654329, AA575854, AI568346, AI926112, AI565406, AA193247, AI689674, BG253912, AI619781, AA931604, AI630615, AI769728, AB048935, S77771, Y14485, AF114170, AF130470, AL049382, AF081366, S69385, AF093542, AF247674, AK026784, L00972, AL096751, AC002467, AC002401, AF110520, AP000839, AP000839, AP002833, and AP002833. HTEIP36 23 520468 1-738 15-752 AI695417, and AC037454. HYBAY77 24 520394 1-801 15-815 U49973, AC006080, AL353802, AF031076, AF031078, AF030876, AL022318, AF178030, AC006398, AL034410, AL049176, AC007380, AC010967, and AL031848. HROAE78 25 520365 1-864 15-878 AC022509. HSAVP17 26 519846 1-836 15-850 AV758870, AI282479, AW855643, AW275432, BE148969, BF946053, BE138594, AW974932, AA704393, AW021917, AA630854, BE062478, AI361090, AV760915, AA456924, AV760508, AA536040, BE148935, BF675251, AV761107, AA829036, AI609972, AV760014, BE062476, AW806990, AI278972, AW271904, AV695478, AW502237, AW970958, BF681619, AA598927, AA757661, AI888468, AI978782, AW969941, AW851405, AI362442, BE138484, AL119247, BF991208, AV756491, AA524616, AW026305, AI421950, BF772474, AI419337, BF811714, BE062159, T74524, AW327624, BE062484, AW383952, BF589824, AW958962, AL037714, AW272294, AA468131, AI635028, BF804359, AI358712, BE178231, AV754716, AA558404, AI380617, AI912401, AC005086, AC005527, AF047825, AC004964, AC000025, AC005103, AC005529, AC007383, AC008747, AL137818, AP002028, AC002429, AL159168, AC005082, AL139095, AC011452, AL117381, AC002115, AC008812, AP002085, AC000035, AL137800, AL135927, AC007227, AL121653, AC001231, AC004876, AC005995, AC002418, AC004778, AL031589, AL139100, AC007193, AC006967, AL021918, AL136981, AJ400877, AC007263, AL121895, AC007395, AL035462, AC016526, AP000193, AL355392, AC005080, AC003029, AC002119, AL117356, AL161445, AL031311, AL121897, AL135744, AP000117, AL121753, AC011465, AC008102, AC007707, AC004522, AC023510, AC010412, AC009004, AC006241, AC018712, AL049766, AC003043, AC005755, AC007912, AP001718, AP000313, AP001748, AC005257, AC002996, AC020898, AL009179, AL133174, AL133289, AC021016, AC025435, AC010627, AP000050, AC002369, AC004386, AC004966, AC005933, AF109907, AC010422, AL160191, AL133545, AC004865, AL121890, AC024078, Z82172, AC005545, AL158040, AC004253, AC005924, AB045146, AC008745, AC004847, AL049699, AC021999, Z95331, AL022316, AC005088, AL049569, AC020754, AP000501, AC008379, AL121920, AC007055, AL034549, AC020893, AL133245, AC006077, AC006071, AC018639, AC007386, AL096701, AL138756, AC009470, AP001631, AC004148, AC005400, AC002364, AC002425, AL022326, AC005236, AC002553, AC005620, AL035086, AC008651, AL133332, AC009086, Z83844, AL022323, AC003006, AL135902, AC003101, AC008641, AL034429, Z85986, AC025588, AC007283, AC005057, AC020917, AC005519, AC074338, AC021036, AC027319, AC005480, AL136137, AL138878, AF207550, AC010458, AC002314, AC015853, AC004089, AC008403, AC006277, AC016995, AL159158, AC000069, AF217403, AC005696, AC006117, AL009181, AL354864, AC004989, AL021397, AF307337, AC008753, AL008718, AL022163, AL109976, AC004560, AL121601, AC011508, AC005004, AL357153, AC009244, AC004686, AC007956, AC005971, AC083863, AC002350, AC004884, AC002404, AC002549, AL136972, AC005058, AL035423, AC005594, AL049653, AC011464, Z93015, AC005701, AC005081, AF001552, AC008521, AL024498, AC024084, AL078645, AL136365, AL133294, AC004659, L31848, AL022320, AJ133269, AC004662, AC005666, AP001728, AC007450, AC003950, Z75887, AC002504, AL031588, AL136418, AP000350, AP000547, AL138735, AC008616, AL122001, AC022516, AF124730, AF001549, AL049757, AC008738, AC007066, AC008736, AP000557, AL035454, AC007326, AC004819, AL049748, AL008582, AC004099, AL033527, AL031282, AC005940, AL138976, AC011742, AL008721, AC010633, AC010633, and AC010633. HSIEA14 27 520387 1-774 15-788 AW301736, AL042667, AL042670, AV695478, AW328202, AW238341, AI538491, AA569494, AA572983, W42588, AI278972, AL120141, AI821382, BE042006, AI307201, AA501461, AA837597, AA514450, AV761433, AI753969, AA613761, H68343, BE139139, AI250552, AV648653, AI284543, AW020612, AI689198, AV656063, AW613448, AI005613, BF917473, AI254770, AI744830, AI820534, BG030789, AI251284, AI251034, AI251203, AI223626, AI249853, AA526099, AW023515, BE138387, AA602105, AI798407, AI439393, AI690497, BE169870, AI692245, AL133519, Z83819, AC002350, AC021036, AL031733, AP001706, AC005844, AF017104, AL157372, AL117341, AP000073, Z84487, AC007282, AC011742, AL022336, AC019106, AC008011, AF049895, AL109797, AP001710, AL160231, AP001759, AL137073, AL391114, AL049713, AL133454, AP001683, AC058791, AP000104, AC007880, Z93341, AC011904, AP001714, AC008738, AC016689, AC008543, AC009225, AL024507, AB000882, AC006449, U96629, AP000344, AC007225, Z98946, AL050333, AL049838, AL121782, AC004491, AL009181, AL035249, AC005064, AL121825, AF205588, AC024082, AC009060, AC005663, AL121949, AC007546, AF107885, AP002530, AC011500, AL137060, AC005969, AC005216, AL031848, AC005291, AL133453, AL353802, AL078591, Z98742, AC002418, AC004386, AP001728, AC013429, AC018513, Z83843, AC002303, AL139826, AC005703, AC008008, AC003042, AL033381, AC005332, AC007938, AL121872, AC006121, AL138755, AC073574, AC011311, AL139109, AE000658, AP001725, AL138828, AC009236, AF196971, AC002429, AC009016, AC012156, AL157791, AC004847, AL109743, AC006160, AP001712, AC007486, AL157413, AC005352, AL109923, AP000692, AC005386, AC004977, AL034550, Z96050, AL360227, AC000004, AJ229041, AL121878, AC008267, AC025442, AL117333, AL136979, AL050317, AC008750, AL359704, AL031681, AF027390, AC004743, AL008732, AC009501, AC007690, AC005562, AC078889, AL355886, AC009238, AC005214, AF280107, AL121852, AC009470, AC007773, AP000352, AL031685, AL136365, AC005821, AP001720, AC020898, AL356379, AL049760, AL049776, AL441887, AC010219, AC003992, AL138752, AC012351, AC012309, AL080317, AC002381, AL035458, AL031289, AC003662, AC036103, AC007312, AL445687, AC006120, AC024600, AF241726, AC005358, U80017, AC004859, AL133153, AC027689, AC005069, AC007376, AC009953, AC018684, AC010363, AC007868, AC004833, AC004000, AL031427, AL022318, AC007240, AC003108, AC010328, AL132777, AC007542, AC006455, AL157827, AC006001, AC007637, AC007262, AF015720, AC006996, U91323, AL158167, AC004745, AC074331, AL121585, AC006372, AC009455, AF190464, AL035555, AC022493, AC003029, Z84474, AC016543, AC027644, AP001660, AC008518, AL391259, AL049834, AL121652, AJ239323, AL163853, AC002289, AC005488, AC004887, AC005690, AC007000, Z98950, AL109655, AL133519, and AL133519. HSNAQ47 28 847453 1-824 15-838 BG031135, BE882847, AA508130, BF897342, N84247, BF793144, H56425, BF735575, BF735202, BE734983, BF735512, BF735472, BF735739, BF735568, AI281168, BE543895, R86099, BF735124, H25599, BF991203, H44341, R70461, AI273738, BE830524, BF735219, BF734982, AI221561, AB007962, AC018593, AC018593, AC025992, AC025992, AL162612, AL162612, AC024468, and AC024468. HODDN65 29 520348 1-741 15-755 BF526964, AV734149, AV760019, AI246796, BE063437, AL135377, AI061313, AA515048, AW274191, AI306232, AL046519, AI251576, AI248050, BF828714, AI311647, AW973992, BF826830, AI207465, AW505253, BF340002, AA303007, AW243793, AA704393, T05118, AI583466, AA504818, T74524, AW855643, AW468048, AA737309, AW732205, AI270177, BF821897, AV755654, AW504168, BG222813, AW965008, AI085242, AW516080, AW500684, AL079734, BE077105, AI380617, AI499954, BE062478, AW237905, AA806804, AA484201, BE148969, AV703187, AI612142, BF724699, AA513551, AA730305, AA515728, AW970940, AI491755, BE301584, AW963444, AI192440, AI053827, AV741663, AA524616, AA515723, AW975626, AA827383, AA678950, BF447461, AW963463, BE138594, AA484366, AI610941, AA829036, BF811714, AW407632, AA569089, AI583252, AA502532, AW502873, BF990660, AW969941, AL046471, BF821009, AC006111, AP000553, AC004644, AC005225, AC005484, AL354889, AC006483, AC005229, AL138759, AC008102, Z84468, AC010202, AL022311, L78810, AC004851, AL008725, AC015550, AF006501, AP000075, AL022165, AC010326, AC007151, AL031685, AC006974, AC011530, AC005821, AC011462, Z97876, AL049835, AL122001, AC004448, AC004686, AC009247, AC006388, AC009311, AC004821, Z99716, AC005480, Z86090, Z98044, AL133387, AL035462, AL049569, AC007249, AC010363, AC002400, AL109930, AL121675, AC008641, AL121845, U63630, AC002312, AJ003147, AC004234, AL035086, AC008072, AL031587, AC007620, AL109963, AL133286, AL049766, AC006077, AL022238, AL137139, AF107885, AP002085, AL033529, AF254822, AC005793, AC004000, AC006480, AL445248, AC005089, D89013, AL034369, AC005071, AC011442, AC005940, AC008736, AL353701, AC004382, Z93015, AP001330, AC006312, AL034420, AP001748, AC005015, AL034424, AC006512, AP001629, AF001552, U08988, AL031847, AL136300, AC005972, AC007739, AL023807, Z82215, AC011490, AL118501, AL022069, AP000065, AP001752, AC004971, AC006344, AC005098, AC000379, AC011811, AC004895, AL034380, AC004832, AC002352, AC008760, AC010150, AL034421, AC004876, AL109936, AC015853, AC012384, AP001759, AC010519, AL163208, AC005971, AL031282, AC009086, AC007276, AL137072, AC008066, AC004622, AL133480, L78833, AL020993, AC004542, AC004494, Z83838, AC006549, AL109976, AC006262, AL022336, AC004966, AL022322, AC006057, AC007384, AL161659, AP001631, AP000555, AC008745, AL031727, AC008115, Z93023, AL096701, AL080317, AC004477, AL122020, AC010524, AL121899, AJ009616, AL034562, AP001435, Z93241, AC005358, AC004867, Z82244, AC004797, AL121753, Z98949, AC005377, AC004143, AF190465, AC019155, AC016025, AL135901, AP001754, AP001728, AC005488, AC003963, AC005602, AL133553, AC006449, AL035252, AP001610, AP002535, Z93244, AC002563, AL133174, AL034417, AC011495, AC009516, AC007676, AC004084, AF104334, and AK023410. HSVAM10 34 520328 1-419 15-433 AI654853, AC021164, AC021164, and AC021164. HSNBN57 35 526721 1-628 15-642 AA505101, AC007263, AL157736, AL357093, and AC007263. HSVBD22 36 520557 1-653 15-667 AL138996, AL138996, and AL138996. HSAWA27 37 520302 1-640 15-654 AW021917, AI753488, AI040051, AI499954, AA809546, AA584489, AW969743, AW516304, AW270385, AI679413, C06046, AA483606, AW338021, AW500250, AI753113, AA570740, AA315361, AW963444, AW243793, AA070899, AW105729, AW083934, AA613627, AV649853, W95976, AW341903, C75406, AW167154, BF764092, AA568204, C75574, AI598003, BF871805, BF673854, AI004591, AI002952, AA568314, AA610433, BF850062, AA491821, AA488746, H18120, AI702049, AW068316, BF030278, AI590499, AW957372, T50676, BE019467, AA878140, AI754955, AP001707, AC004707, AC005046, M37468, AJ277546, AC004846, AC008482, AL121656, AC000025, Z99716, AP001760, AP000042, AP000110, AP001716, AP000338, AC005202, AC005969, AL136124, AP000216, AL354864, AC005924, AC006329, AL031846, AC027319, AC005913, AP001714, X02571, M12901, AC004386, AC008556, AL022336, AC026776, AL008735, AC005291, M91159, AC005778, AC004236, AL157760, AP000104, AL135927, AC007227, AL118520, AC005840, AC005000, AC004000, AC011742, AC006511, AC004971, AC008403, Z83826, AC008848, AC010328, AL035420, AC007383, AC004686, AF069291, AL022237, AL132659, AC004887, AL031286, AJ009615, AC018644, AC022148, AL031680, AP000555, AL117381, AC011490, AC005881, AC004531, AC005755, AL078584, AP000556, Z99128, AP000557, AC004840, AC012309, AC008072, AC007537, AL365505, AC004859, AP000289, AC007919, AC020904, AL136418, AP002015, AC007488, AL049830, AL161665, AC023490, AL050341, AC005971, AC007051, L78833, AC002301, AL049872, AC005074, AC007597, AC002369, AC005071, AL353602, AC010789, AC015651, AC003029, AC004650, AC000003, Z81450, AC004929, AC005280, AL121655, AC006345, AL158830, AC004476, AL135783, Z98172, AL050335, AP000692, AP001666, AL121897, AP001712, AL353194, AC006126, AC004841, AC010422, AC008068, AL078590, AP001725, AC002418, AC006468, X74984, AC008747, AC008521, AL031255, L78810, AL133391, AC000159, AC007731, AL031284, Z98884, AC005500, AC018738, AC005839, AL021391, AC008784, AC009086, AC025588, AL353802, AC006459, AC016395, AC000379, AC004263, AL122001, AL033520, AC006130, AL133448, AC006441, AC006038, AC006484, AC004966, AF215937, AC005730, AC006057, AP000553, AC005180, AC008745, AL035071, AC002985, AC004975, AL109627, AC007957, AL050308, AL096861, AC004491, AL031587, AC005703, AC007421, AL022316, AC005598, AC011495, AC068658, AD000671, AP000512, AF023268, AC040171, AC010618, AC005484, AC005821, AL133245, U78027, U80017, AC007201, AP001710, AC005529, AC022243, AC022243, AL358353, AL358073, and AL358073. HSFAH43 38 520399 1-717 15-731 AL038713, AI818151, AL133942, BE728951, AW157413, AA085707, AF074627, AU146974, AI925647, AL044349, AU145383, AI367384, AI887321, AF063514, BE549207, AA902828, AV719347, AI685116, BE646447, AU122382, AV726924, AL041411, BE409924, AU121228, AW847204, BF032731, BF681274, AU157470, BE385675, AI559442, AA864823, AF074693, AW852540, AW084901, AA767353, AW852539, AL133889, AI264673, AI858607, AL534431, AW139132, AA659014, AI610776, BE879973, BE165748, AI568919, BG059728, AU148220, AA745961, BF839073, BE893036, BE161913, AI924175, BE065468, AW177120, AW085676, AU143935, BF802808, AA581433, AI819528, AI250812, AI114529, BE065692, W58442, BF744176, BE745970, AI189033, BF953914, AI817158, AA932087, BE155605, BE065645, BE176566, BE065599, AA493735, BF760188, BF819519, BE179562, AI147839, AI749571, AI827133, AI675848, AA130536, AA055654, BE179906, AI132962, AW963409, BF760502, N24958, BE079382, AI376984, AL119355, BE267153, BG169215, AA493998, AI499286, AL523955, BG059067, AI733728, AA130476, AI453790, AA778304, BE079397, AW177237, AA174085, AA663566, BF963854, BF967102, AW074001, AW813858, AW795421, AI082077, BF869662, AW089655, AA846188, AA189081, AI133073, AW262471, BG010863, AW419031, AI862874, AA287329, W85828, BF806618, BE065644, AI973178, BE145271, AW177226, T16214, BE044603, BF854640, AA129986, AF063501, AI051341, AW995608, AW938300, R80440, BF576607, BF056069, BF915223, AW090210, N64574, AL521095, AI439860, BF878385, AV732745, BG235936, BE179619, AW177231, AW177317, W49501, AW102963, BE065597, BE154392, AW947245, BF935745, BE155635, AI610326, AI811854, AA807609, AA843874, T57073, AW571697, AW962610, AI984510, AA157033, AW440317, AI246569, AI027421, BF689260, AW813744, N26540, BG230953, AI921101, AW167452, AI983921, BF339110, AV764113, AI927861, AA121916, AA099788, AV661095, AW235478, AW847202, AW023397, H97952, AW847178, AA092309, AW151307, AU145663, AU118990, AA501873, AA984116, BF943200, AW945630, AA709024, AW889871, BF958183, AI557354, AU144339, BF751284, BF888049, AI832184, AW177266, AI952804, AI862212, BF843702, AI479035, N76274, AV729448, AW827404, AW517766, AA911409, AW272376, BF821660, AA811111, AI025602, AW833263, AA854527, BF946785, AV730063, H20876, BE154996, AA889273, BE538852, AI439415, AU143906, AI369914, AI561208, AW948678, AI538654, T62931, AI811494, AA581340, BF946782, AW193992, AA515372, N20521, BF760191, AW833302, AW948681, AA166854, AI346802, BF873305, N90055, AW168798, AW948679, AL139348, U73465, AC046130, AL021408, AL355305, AC068130, AC021070, AL157915, AL391071, Z82193, AL136080, AC008041, AL133370, AC009481, AL031114, AC005740, AL161743, AC021998, AC019100, AL121933, AL035453, AL078601, AL034408, AL078644, AB045365, AL121882, AL132826, AC026201, AL353745, Z93015, AL035689, AL133417, AF241733, AC000112, AC008929, AC002066, AF241726, AL356596, AL031684, AJ133269, AL157775, AC005341, AC005000, AC016607, AL033379, AC013458, AC018360, AC007514, AC009501, AL049734, AL050309, AC006600, AC003686, AL008627, AP002535, AL049778, AC007251, AC004385, AL354891, AL139093, AL135978, AC011478, AC007744, AL358214, AC005146, AP000173, AL163194, AC008572, AL121932, AC001608, AL157694, AL109750, AC008116, AL109759, AL138773, AC060232, AL139232, AL161730, AL138696, AX015914, Z92542, AL109800, AC002519, AL121879, AL079303, AL024507, AL390965, AC063956, AF130249, AL023806, AC002402, AC018468, AL158812, AC017067, AC018504, AC026184, AC008818, AC023118, AC068313, AC018492, AL109620, AL135940, AC061958, AC009241, AC008134, AL139009, AF241727, AL133381, AC067744, AL356750, AL133409, AL138810, AJ298105, AL033403, AC060834, AC023430, AF207550, AC008583, AP001721, AC004103, AJ277546, AC022274, AC003668, AC006226, AC007000, AL139350, AL133284, U69729, AL031655, AF307337, AL022577, AP000333, AL356982, AL034396, AL035464, AC009289, AL122016, AC007276, AL158141, AL110505, AC010738, AC005191, AC011230, AC083875, AL136970, AC005284, AC006516, AC068799, AL161788, AC006556, AC004384, AC004388, AC023478, AP002026, AC023483, AL163206, AL161916, AC008861, AL133462, AL009172, AL365214, AC006313, AC008598, AL360088, AC034195, AC007570, AC024947, Z95437, AP001342, AL391260, AC020717, AC006010, AL035700, AL139109, AF238375, AC005164, AL078615, AL022394, AP001821, AL157830, AC010276, AC011507, AL450448, AC007358, AC003051, AC024900, AL163152, AC024600, AC005213, AC006581, AC073102, AF027390, AP000068, AL157777, AL359703, AC004111, AL139014, AC002379, AL392106, AL359854, AC005214, AL356800, AF130343, AC007486, AL133514, AL450169, AC006352, AC022428, AC006143, AF238380, AP001677, AC007392, AP000112, Z95400, AL133411, AL034407, AL022171, AL133247, AC000120, AC009401, AL392108, AL158040, AL158015, Z82216, AC006984, AP001538, AL109804, AC008174, AC021863, AC009948, Z72001, AP001660, AL031653, AL109809, AL355540, AL031075, AC005019, AL354943, AC026197, AC005138, AL137012, AC022360, AL139813, AC012519, AC005358, AL034375, AP000340, AC011594, Z97987, AC004848, AL031726, AL031726, AC023412, AC058811, AC008041, AC008041, AC011739, AL356513, AL356513, AL109804, AC006226, AL079303, AC010192, AC010192, AC069443, AC069443, AC069443, AC006357, AC006357, AC026494, AC026494, AL121879, AC008134, AC008134, AL022165, AL022165, AL022165, AC021329, AC027116, AC027116, AL022395, AL022395, AL022395, AC005000, AP000445, AP000445, AP000445, AF207550, AF207550, AL138810, AL138810, AC007436, AF011889, AC005740, AC005740, AC004958, AC024947, AL031601, AL031601, AC007570, AC007570, AC013708, AC013708, AL157762, AL157762, AC007338, AC007338, AC018845, AC018845, AC068139, AL022394, AL022394, AL162390, AL162390, AL162390, AC011086, AC025802, AC025802, AC025802, AC069153, AC069153, AC007623, AC007623, AC016119, AC016119, AC018494, AC073487, AC073487, AL139109, AC025624, AC025624, AC025624, AL355075, AL355075, AC011435, AC011435, AC012321, AL355305, AL355305, AC009266, AC000120, AC000120, AC002060, AC002060, AC003075, AC006084, AC006084, AC006084, AC006144, AC006144, AC007000, AC007000, AC007000, AC007276, AC009501, AC009948, AC009948, AC009948, AL022069, AL022069, AL049875, AL078644, AL078644, AL110502, AL110502, AL133304, AC037472, AC037472, AC018568, AC018568, AC011432, AC011432, AC011432, AC021652, AC021652, AC021652, AC063920, AC063920, AC063920, AL133124, AL133124, AL133124, AP000974, AP000974, AC022194, AC022194, AC022194, AP001645, AC027552, AC027552, AC012036, AC012036, AC022845, AC022845, AC012447, AC012447, AC012207, AC012207, AC002379, AC002379, AC002379, AC006556, Z99758, AC007345, AC007345, AC010210, AC010210, AL356982, AC011153, AL355474, AC005023, AL031387, AL133370, AL133370, AL133370, AL354751, AL354751, AL354751, AC011123, AC011123, AC011123, AC018840, AC018840, AL355489, AL157899, AL157899, AP001562, AP001562, AP001562, AC025508, AC025508, AC011768, AC011768, AC005146, AC006984, AC006984, AC007358, AC011327, AC011327, AL031650, AL033403, AL033403, AL034408, AL034408, AC074245, AC074245, AC010798, AC020656, AL138682, AL138682, AC073118, AC073118, AC012600, AC012600, AC012600, AL138814, AL138814, AB045359, AC005090, AC005164, AC022078, AC022078, AF130343, AF130343, AL021408, AL033522, AL035689, AL035689, AL035689, AL035700, AL109628, AL157915, AL157915, AL157915, AL353812, AL354833, AL354833, AL355052, AL356802, AL365475, Z95400, AC010736, AC010736, AC023965, AC011401, AC011401, AL121933, AL121933, AL359754, AL359754, AL159175, AF027390, AC011823, AC011823, AC007849, AC007849, AL024507, AL024507, AL391071, AL391071, Z92542, Z92542, AL161645, AL161645, AL161645, AC022122, AC025555, AC006313, AC009289, AP000068, AL135978, AL132826, AL132826, AL158158, AL158158, AC021850, AL136327, AL136327, AC078802, AC078802, AC074250, AC068393, AL354708, AL354708, AL354708, AL359271, AL359271, AL356259, AC003099, AC005166, AC006195, AC007743, AP001821, AP000598, AP000598, AP001567, AP001567, AC023109, AL133385, AL139136, AC021201, AC007744, AL133381, AC015521, AC015521, AP001321, AP000409, AP000409, AC023555, AC008583, AC008583, AF001905, U69729, U69729, U69729, AC069525, AC069525, and AC015464. HSPAA60 39 526447 1-364 15-378 AI912648, AW387635, BF350647, Z98044, and Z98044. HFAEF57 40 534142 1-628 15-642 AV655597, AW967329, AW963498, AV706016, AW966767, and AL121984. HEGAH43 41 532596 1-428 15-442 AA400429, AA994981, AA846419, AA453384, AI015471, AA992965, AA400538, AL360078, and AF327147. HAGDG59 42 534165 1-1720 15-1734 AV694248, BE895909, BE903848, BG027942, AV651246, BG109867, BF240140, BF217526, BF669125, BE779936, AV650099, BF971092, AW875350, AW956342, BF107182, BF697022, BG166672, BF030619, BE881774, BE548671, BF247518, AI888053, BF667451, BE872808, AI768748, BF792803, N37046, N23484, BE872350, BF239058, AW664126, BF107464, W88681, AW338066, AW952476, AW402833, BE971415, AW853145, BF968304, AI636324, N24759, BF665132, BF213364, AA830565, AV697089, AA167203, AW023148, AI815125, AI685119, H98763, BE465545, AW853521, AW405572, AA481430, AW604402, AA481434, AA223067, AA902413, AW578436, BG258700, AI954984, AA045833, AI567716, BE856103, AA577610, H42133, AA553538, AA470843, AW467047, AW169016, AI961753, H38614, AA835545, AI262411, AW192401, AI193508, AA576473, AV660930, AW262909, AA263040, BE927225, AA481670, BE782154, W88620, AA394254, BG261374, AA730743, AA653560, R80477, AV651327, BF572366, AW193089, BF904780, AW470979, BF904779, AW295546, AA045967, AA329460, H42134, AA213836, R23904, BE243520, BE693582, AI263974, AW293723, AA481674, H61494, BF674778, R23903, R80672, BF032805, R61171, N79745, BF382094, H38856, AA328661, BF902314, BF243422, R27506, BF894060, T72506, AW361405, H62468, W07107, AI468319, AA362581, R27793, AW853797, AW337877, AW075817, BF031795, BE866675, BF905580, T82403, R27885, AW339053, AA377009, BF791479, AI708354, AA485696, BF239244, AW051074, BE866177, AI497897, AA485827, AW273624, BF905573, BF205089, AW022407, AI678575, Z21560, BF238897, BF699925, BE926278, BF239919, AW836245, AF126780, and Z64479. HNGBX63 43 532615 1-503 15-517 AL137077. HE2AG50 44 532595 1-472 15-486 AA278487, AL357314, AL357314, AC009337, and AC026052. HCUIN80 45 534128 1-812 15-826 AW500684, AI755214, AA284247, AI754567, AA535216, AI754105, BF768846, AW576251, AI056177, BF725761, AA410788, AA714011, AW328331, AA485328, AI792521, AV710214, AA582073, AA714110, AA515048, BE138509, BE968744, AA651632, AV738383, AA502991, AI962030, AW023111, AA837686, AA515728, AI792499, AL079734, BE138594, AV756491, AU147162, AW575000, AI053688, BF675251, BF804385, AA503298, AW576326, BF915799, AL037052, N23913, BF725844, AA714288, BF939548, BE062159, AI053793, BF526964, AW069227, AI792575, AA084609, AW973992, AA809546, AL037771, BE066564, AA283730, AA312559, AA659832, AI345132, AI801505, AA176978, BF032064, AV762354, BE301584, BG222813, AI612142, AA630854, AV763026, AV763058, AV760915, AI923052, H07953, AI755038, AI669421, AV763057, AW970940, AW970571, AI742633, AV758067, AW969743, BE739230, AA701080, AW237905, T05118, BF854308, BE501593, AW798093, BF839876, AV655282, AA601278, AV702109, H58354, BF771774, BF527070, AA595661, AW962651, AA128511, AW819125, AA019973, AI754653, AW467607, AW965807, AA526326, BF916767, AA593537, AA523792, AV759199, AW468009, AW576034, AV742957, AI440117, AV760508, AA832145, BG250286, AA492584, N95817, BE896991, AA618316, AV763410, AV753973, AW816516, AA829036, AV653153, AI583252, BE877891, BG059972, BE049032, AV762633, BF681619, AI554471, AI311779, AA536040, T74524, AA526625, AC004020, AL121985, AC068999, AC007687, AL034548, AC005529, AC005034, AL353898, Z82205, AL355392, Z85996, AC011290, AC023051, AC000025, AC005527, AC003013, AF003528, AC022073, Z94056, Z84469, AC011495, AC006389, AC002300, AC009024, AF001549, AC006948, AC005971, AP000014, AC007934, AC012005, AC005823, AC007590, AL161731, AC006387, Z98304, AL445483, AC004832, AL035685, AC005627, AL358293, AC006460, AC003051, AL078581, AP001729, AL096800, AC010170, AL356240, AC005792, AC004831, AC006236, AF196970, AF168787, AP001436, AC034242, Z93023, AL356652, AC010305, AL121601, AL050317, AC007333, AC007358, AC010605, AP000156, AP000070, AC003956, AC007382, AL121891, AL121747, AC004629, AC002301, AC005207, AL137061, AC007251, L11910, AC008985, AC005740, AC018633, AC006538, AL078461, AC012309, AC007488, AC002310, AC002492, U73169, AC005338, AL022162, AC007221, AC006449, AC008379, AC002128, AC006028, Z84484, AL139099, AP000512, AL035467, AF001548, AC005324, Z83822, AC004103, AL031733, AC007387, AC006023, AF195953, U75931, AL139396, AC004156, AL117348, AB023049, AC002306, AF124731, AC009363, AL080242, AL049631, AL035587, AC008745, AC007160, AC006333, Z97196, AC011465, AC004686, AC002550, AL109976, AC007653, AC004804, AL162424, AC005048, AC002302, AC007690, AC002319, AL049712, AL139289, AC005726, AC002430, AL049634, AL137145, AL080243, AC004253, AL136137, AL136102, AP001725, AP000103, Z95114, AC006544, Z92844, AX000035, AC005081, AL035684, AB026899, AC007056, AC007384, AD000685, AC005821, AC003110, AC006120, AL096755, AP001753, AC012081, AC009333, AL035671, AL034420, AL008719, AC011452, AC011479, AD000684, AL031311, AC005751, AP001675, AC008266, AL049569, AL109805, AC008784, AL031005, Z68870, AC008812, AL450226, AF003627, AC002369, AC004025, AC004595, AL137039, AL121900, AC011445, AC005274, AC027312, AC008395, AL139384, AL138976, AC009516, AL035086, AL161941, AC009244, AL031289, AL137162, AL022328, AC007371, AC000029, AL117332, AL049697, J00083, AL139343, AC005220, AC002347, AC005952, AC004928, Z98752, AL031291, AC004796, Z96811, AP000553, AC009509, AC018712, AC003668, AC005369, AC020637, AL079304, AL118505, AC006511, AL163249, AC008928, AC002544, AC021016, AC007919, AL121572, AC020552, AL138741, AC022148, AC004089, AF001550, AL133545, AC005200, AL136304, AL050335, AL050349, AC006271, AF165926, AC003667, AL133174, AL133153, AC011469, AC006000, AC008403, AC008623, AC010677, AC022143, AL133382, AL135818, AL163279, and AC008465. HADCL29 46 532056 1-680 15-694 H03258, AI299317, N52545, AC025153, AC025153, and AC025153. HAPPS89 47 532135 1-842 15-856 AI963720, AL046409, AV728425, AV710066, AF330238, AI270117, AV725423, AI334443, BF337291, AV733830, AV760042, AI431303, BF697673, BF241967, AI305766, BF677892, AI284640, AA490183, BF668217, AW193265, AI754658, AI613280, AV762098, BG249643, AV734666, BE276880, AV760777, AV764307, AI345654, AV762395, AV762535, AV759172, BF679304, AW963497, AW274349, AI350211, AV761362, AL138455, AW953071, AL044940, AL138265, AV763670, AV762064, BE049139, AV764241, AV740801, AA551503, AV764329, AV760057, BE276641, BF680074, AW438643, AW419262, AV763540, AW265385, AV763971, AI289067, AV761843, AW975425, AL042853, AF074677, AL037683, AL041690, AL039958, AI619997, AV761489, AW979060, AV760937, BG107801, AI688846, BE350475, AV761106, AI341664, AV759204, AW276827, AV764578, AW193432, AA244357, AI061334, AV718260, AL119691, AI754955, AV762505, AV760774, AV763354, AW965008, AV762645, AW303196, AI375710, AI281881, AV759437, AI133164, AV762959, AV729881, AV763449, AL045053, AU147104, AW083402, AW502975, AA483223, BG109996, AV759382, AW833862, BE150580, AW301350, AV763847, AI814735, AV760466, AA584201, BF216716, AW327868, BE895987, AL042420, AV725431, AW974109, AV702857, AV762139, AU151000, AV762111, AA491831, AV759505, BE253048, BF541120, AV732891, AI801482, BF854876, AI345681, BG222267, AI345675, BF827410, AI708009, BF311000, AA533333, AA610491, AV735370, AA587604, AA581903, BF965007, BF991286, AW406447, AV762050, BF939954, AI053672, AI064864, AI610920, AW975049, AI340453, AA470969, AV713243, BG059450, AA584167, AV759507, AI281903, BE967369, AV761745, AV718479, BF475381, AI344844, AA682912, AV763633, AL046205, AA631507, AV682003, AV719506, AA719805, BF679256, AV763255, AI744995, AV762015, AV735495, AV762067, AW407578, AI355206, BF684828, AV764530, AA649642, AA577906, AA623002, AV759274, AW503666, AI679782, AW500125, AV761786, AI307608, AW517388, AV658733, BF130605, AI732865, AW302013, AV742057, AI349850, AI559705, AW410400, AI076616, AV760039, AI307201, AV760106, AA664015, AW972312, AW517377, BF724372, AA680243, AW518220, BF679274, AW472872, BE139146, AW021583, BF681576, AV710774, AV759518, F36273, AW576391, BF691714, AW970896, AV763122, AV719422, AW083364, AV759329, AA720702, AV756693, AA829223, BG036665, BF793766, AI634384, AL041412, BG059314, AV760700, AV735239, AW408717, AI305547, AI471481, AV762009, AL120687, AW969629, AI799642, BF680041, H08791, AA525824, AW513362, AI133102, AW970848, AW995093, BF964720, AA491814, N53150, AP001718, AL137881, AC023275, AC007919, AP002026, AC008770, AL139388, AC005516, AP001675, AL096840, AL078590, AL117382, AL035411, AC004948, AC007404, AC005046, AP000501, AL157915, U18394, AF015149, AC002470, AL035665, AL445604, AC006989, U18391, AF302689, AL137141, AC004638, AL031650, U18395, X55925, AL353739, AC003080, AC004832, U57009, AC010088, X55926, AF265340, AL139009, AE000661, AC008279, AL353807, AC009087, AC004840, AC011495, AC008602, AC008372, AC006501, AL391867, AC025735, U85198, AL022302, X54180, AC010104, AC009244, AC008562, AC005666, AC005323, X54181, I51997, AL355497, AC006338, AF015148, AL138759, AL445223, AC007243, AF015151, AC083871, X54175, AC007620, AC006376, U95742, AC005393, AC004019, AC005019, U67221, AC022274, AL096776, X55931, AC007216, AL163853, AC004765, AF029308, AC007274, U18392, U02531, AC010879, AC007298, AC003003, AL161666, AP000855, AL360227, AL160237, U67231, AC005859, AC027319, X54176, AC006539, AC004987, AC008733, U63630, X54178, X53550, AC004816, AF217490, AF015156, AL354720, AC022013, AL133517, AC002400, AL135796, AC004848, Z93241, X55924, AC009223, AC009506, U18398, AC022027, AC006153, AL161892, AC004884, AC005244, Z99495, U18387, U57005, AC087093, AC068713, AF015147, AC002094, AC011310, AL096829, AC005699, U18396, AC020893, AP000548, AC005250, Z84814, U18399, AL033383, AL049762, AC006251, AF015157, AC006328, AL022323, AC004453, D83989, AL353696, AL121748, AC002377, U57006, AL135839, AL096863, AC023344, AC006925, U67233, U57008, Z49816, AC009470, AC010168, AC027689, AC005914, AC005923, AC006999, AL031660, AL020995, AL118520, AC004814, AC019187, AC027644, AF067844, AC009482, AC024952, AC007350, AC003977, AC006213, AC073893, AP001714, AL137228, AF010238, AC001231, AL078461, AL022163, AL356750, AL136418, AC020637, AC008534, AC004953, AL357141, AC002128, AC010726, AC021878, AC007364, AC010384, AL031432, AL359077, AL163247, AL158196, AC016995, AL161724, AC008249, AC022432, AL035683, AC010723, AC011749, AC004825, AL139352, X55927, AL109853, AC007011, AC004989, AL136372, U66059, X88791, X54179, AL138715, AP000117, AC003007, AL136081, AL355385, AL021406, AL390168, AC005988, AL035659, AL136173, AC018507, AC018758, AL121959, AL133480, AC009948, AL049874, AC006450, AL049868, AC009179, AL121751, AC010682, AC007363, Z98742, AL356288, AL050097, AC004808, AL035404, AC006983, AC005387, AC026398, AL157372, AP001687, AC005216, AC005257, U67801, AC004502, AC020873, AC020873, AC027442, AC022766, AL139131, AC073219, AL354696, AC023864, AC023864, AL355975, AC022795, AC068289, AL353659, AC010073, AL353577, AC015587, AC025975, AC072051, AC073849, AC025869, AC055861, AC010853, AC011036, AC016319, AC068519, AC027234, AC015604, AC021212, AC034243, AC068755, AL157775, AL356423, AC034137, AC068682, AC021164, AC009858, AC009858, AC067779, AC067779, AC024475, AC026936, AC073446, AC073446, AC041025, AC023309, AC067907, AP001992, AC016538, AC027584, AC027584, AC027408, AC027772, AC012461, AP001929, AC009095, AC009899, AC023672, AC055788, AL358492, AC024337, AL353139, AC060817, AC011233, AC019092, AC019092, AC022051, AC023968, AC021921, AC021901, AC068196, AC068196, AL161634, AL354657, AC073909, AC013758, AC021401, AL390917, AL390917, AC007459, AC002369, AC002369, AC036208, AC036208, AC009407, and AC009407. HFGAH44 48 533132 1-1629 15-1643 BG169338, BG023827, BF525720, AW948966, AW968891, AA868896, AW043609, BE503306, BF671249, AA863046, AW051836, AI654795, AI808367, AW972853, AI745196, AA482288, AA421645, W84810, BF029772, AA525503, AA470086, AA946801, BE048921, R67044, AA495896, AA903005, AA482196, AA865021, AA425004, AI075164, AW275813, H23981, AI634761, W84811, R45305, AI378172, AI184932, H22701, BE935481, BE463530, AI613129, AW022383, H95118, H24272, AV710267, AW236030, AW444923, AI015394, R66160, BF512442, R20482, AI864299, AI783459, AI366098, AI696350, AI804999, AI628688, R32232, Z45604, Z41288, AI361717, BF372594, BF370363, BF372593, BE142713, BF370390, BF509363, AA398223, BG257307, AJ276674, AC023120, AC023120, and AC023120. HFIHZ96 49 532046 1-695 15-709 AW079613. HFIUR10 50 532060 1-527 15-541 BF195618, AA191239, AW969824, AA009856, AW019964, AA808036, BE677291, AW973259, AW023662, AV742957, AU146063, AI369580, BG109444, AU153717, AV709074, BG032605, AI357823, AW888719, AL110373, AI832009, AV708388, AV725797, BE150580, AA223512, AV734980, AA402529, AA595661, AW410201, AA683069, AA191418, AI144036, AW474168, BF681348, AI590458, AI590499, F08248, AW302048, AV760508, BE794962, AA665181, H07953, AW971071, AA654781, AV763410, AA749035, BF965290, AI609972, BF676985, AV708385, AW504485, AV762633, AW166808, AA282951, AI860535, AI792575, AA634889, AW302950, AL048060, AI254913, AW875172, AI281689, AA668587, AA084619, BF675051, AI354423, AA832077, AI733129, BF674550, AL041924, BE139451, H73550, AA828853, N39953, AW863393, AV757526, AI859946, AW976008, AW023111, AA747234, AI565084, AV710482, AW814024, AV710045, AW963482, AI355246, AA814925, BE077105, AA653182, AA664521, AW440305, AI054397, AA651639, BF725761, AV758073, H15652, BE280771, AW438542, T74524, AW191063, BF940118, AW968205, AV762973, AA552578, BF965924, BF879045, AI251034, AI251203, AI251284, AW805539, BG236628, BE878259, AI250552, AA632556, BF809041, BG029224, BF868994, AW020736, AF236698, BE139139, AW271904, BF978025, BF681424, AU118374, AV758790, BG110480, AI803809, AV758097, AA574442, AV733434, BE155302, AA644664, AI792521, BE246472, BE901278, AA626825, AI686913, AV706237, BE155299, AW302293, AV702609, AA533123, BE968477, AV738383, BF814446, AI891080, AA516190, AA533040, AI284543, BE273825, AW779609, BF525663, AI380617, BF914419, AL079734, BG166965, AW069227, AL043351, AI267161, AV762870, AV658819, AV709273, AL042735, AA503018, AI973173, AL046746, BE062357, AI963705, T69857, AV730245, BF810071, AW301736, Z97987, AC020913, AL031281, AC007637, AL133456, Z93017, AC087225, Z83840, AF245699, AC010349, AC004106, AC004132, AC008925, AC004990, AC010618, AC006275, AL035405, AC006930, AF156495, AP001732, AL139824, AC003037, AC005162, AL050341, AL034420, AC024075, AL117382, AC008521, AP001039, AC005778, AL049569, AL109914, Z95152, AL163541, AC006367, AC005684, AL117377, AL109828, AL031681, AC007488, AC007425, AC007934, AL078602, AC005038, AC009743, AC006538, AC053467, Z95115, AC010203, AC010150, AC006545, AC006546, AC004970, AP001696, AL390736, AC003035, AC007381, AC006253, AC022173, AC003684, AC009331, AL109823, AC004605, AL035682, AC006270, AC016526, AC003664, AL078634, AL009031, AL137802, AJ251973, AC002326, AL356421, AC006388, AL138721, AC004103, AL139150, AP001718, AL109915, AL117187, AC004616, AC017100, AC006080, AC005027, AC017006, AL049540, AC005522, AP000353, AC008008, AC078843, AL161657, AL031280, AL035696, Z81364, AL157938, AL389883, AL024474, AC008266, AC007784, AC002996, AL354816, AC011449, AP000193, AL022313, AC008379, AC005004, AC005514, AL118501, AL009028, Z86064, AL035249, AC002549, AL109984, AF001549, Z84467, AL034419, AC006204, AL353574, AC006960, AC009079, AC009503, D87009, AC005014, AC005859, AC003662, AL035089, AC005751, AL390738, AL133246, AP000694, AL133355, AC004854, AC006237, AK023233, Z85999, AL022323, AC003046, AL031730, AL160071, Z82206, AF029308, AC009600, AC005498, AL133342, AP000348, AC011465, AC004812, AC007221, AL022316, AL035072, Z97630, AC008551, AC007685, AC005034, AL391602, AC005220, AP000117, AL023883, AL109825, AL137782, AL096800, AF252279, AC005695, AC007620, AL022237, AC006481, AC018812, AL035420, AC004611, Z84487, AC018719, AC010163, AL133344, AC011290, AC007292, AC011895, U63721, AC002352, AC011444, AC005327, AC004595, AC002470, AL008732, AC006460, AL137119, AC005786, AC006211, AF134726, AC010205, U52112, AC007597, AC016026, AF186191, AC005215, AF155238, AL121756, AL137796, AP001597, AL136526, AC006084, AC013434, AC010596, AL138836, AC011450, AC000063, AL354984, AC007201, AC022436, AC005784, AC005479, AC009319, AC005324, AC010489, AL049570, AC006014, AP000335, AJ297357, AC007679, AC004583, AL157877, AL023513, AC006162, AC004381, AF205588, AK023462, Z85986, AP000503, AC004522, AC007773, AC005527, AL035684, AL031847, AC004686, AL137784, AC005320, AL031680, AF001548, AL031597, AL139102, AL138807, AC010126, AC003048, AC008015, Z98036, AB030001, AC004000, and AC002357. HLDNA86 51 535730 1-706 15-720 BG034488, BG120997, BE910635, BF314688, BE794497, BE875593, BF340441, BF306972, AA205661, BF203164, BG027046, AA643840, BF525733, BF982559, BG030710, BF792430, BE387269, AI955852, AA146971, AA553820, AI869318, BE276651, AI832232, AW026472, BF195525, BF058723, BF204545, W72049, AW248507, AI765058, AI765242, AA034035, AI884693, AI127262, W79426, AW872762, AI376148, BE391657, AI765029, AI073710, N63475, AI277854, AI339947, AW024138, BF061732, AW439832, AI694928, AA708126, AA813521, AI147240, AW242703, AI123913, AI498928, BG222313, AI268993, BE313144, AW079287, AI301956, AI311112, AI304651, BE314135, BF435947, AI347608, AI075899, AW839917, H99183, R47883, AI560747, BF969005, BE669425, AI346935, AI808064, BF195494, W46277, N23743, N30038, AW136724, AI342962, AA781466, AI338271, AI090370, AA625788, H81310, AA836303, BF205034, AA709462, AA399114, BE262728, H94295, AW204374, BF315735, AA156864, AI969210, AA041385, AV683814, AA708782, AA704970, AI686517, AW968213, AI038344, AA468399, AI423158, AI597845, BG152852, BF304202, AA741397, AA770182, AI219428, AA298982, W94777, N35718, AI374736, AA868201, AA041191, AI497678, AA974145, AA127044, AA468439, AA478010, AA707176, N36015, AW243034, AI203479, H81366, AI341986, AA513969, AA025410, AW204258, BE266232, AA993215, N59833, AV708035, AA991336, AA704288, AA745913, BE018923, AI291572, AW800090, AW000874, AV687411, BE393296, AI379942, AA033915, R06215, BE260130, AW404352, BE645626, AA146970, F27351, AA156956, AI357603, H45882, BE295615, AA542901, AA564888, AI357554, AA970265, AA564958, AI761938, AA935742, F30350, BF081714, AI762149, AI869708, AI220792, BF889167, N24466, AA044694, AW188811, AI919010, R47882, BE833225, AW235465, AW090329, AW961121, BE140096, BF591489, AI956005, AA298368, AV687766, AA398056, AI867544, AA298370, AA296786, W76401, AA478165, AI749204, N40747, W79326, AV661716, AA296693, AA298843, BE928658, H94190, AI369692, AI365332, AA029123, N44247, AI566992, AA918262, AA846379, AI269986, BF311814, W95282, AI915437, N94773, AA298624, AA041425, AA297475, N32448, AA125766, AA041429, F30226, AV687884, BE090937, R36822, AA045652, AV687881, BF750936, AA205774, T24460, AA297517, and AV730348. HNGAN75 52 532613 1-965 15-979 AL353810. HCUIO20 53 534220 1-366 15-380 HLTEF12 54 532354 1-2009 15-2023 AL516839, AL515540, AU124773, BE793826, BE799663, BE791935, AI928841, BG177521, AW005472, BG164346, BE794165, BE744549, AL516204, BE383125, AU117173, BE617751, BE906822, BE617248, AA573936, AW470072, BE646485, BE797812, BE909124, BE740662, AI829978, AI339290, BE741031, AL516852, AW166889, AL534961, AI192955, AW780355, AL517045, AW157313, BE894493, BE293842, AW629424, AW025010, AW070803, AI086145, BE512673, AI418163, AI859725, AU137251, BE255112, AI819465, AI816039, AL534736, AW574541, BG236033, AW005461, AL515541, AA595260, BF313175, AI189477, AW512956, AW769006, AW087972, BE279989, BE900347, AW129615, AI963813, AW675506, BG249791, AL516840, AI093998, AW945181, BF974259, AA599151, AA599393, AI080082, AL531455, AI628281, AI189835, AI284394, BG033989, AI128105, AA194432, BE793622, AA181002, AU155416, AA773073, AI625249, AW156965, AI886855, BF317270, AU159778, AW469006, AU151863, BE272720, AI285334, AA668589, BE205937, BE293376, AI554091, AA194582, AA428271, AA724027, BE778798, AA973557, AL120284, AA977213, BE549412, F28501, AI082349, W20116, AA132428, AA314969, AL516205, BG055939, AI754545, BE793245, AA970441, AI128575, BE739619, T08672, AW472905, BE741487, AI186425, AA884851, AI051160, AI183740, BE739157, AL517046, AI475120, AI470142, AA159395, AI446226, AI241304, AA559012, W86231, AA996169, BE265404, AW950509, AA760649, AA115570, AA159298, F22270, F27528, AU155699, AA679907, M86179, T86865, AW974231, H24001, AV749630, AA807991, AW009279, BG163486, AA505630, AI261447, T31711, H20295, C06410, AW574621, BG035018, AI000329, H09218, C05284, AI097554, AW248736, F33051, AA455991, AI654382, AA194706, H09161, AL516853, AA156018, AA132384, BF771643, AA588330, AI240896, AA338059, R98122, N88680, BG105261, AA346227, C03909, AA009594, F17542, AA371843, BF447041, F36262, AU142328, BE044395, F30521, T70010, AA658541, AU129291, AI694006, C03698, T69940, T85208, H80955, AA346119, BG165873, AA847638, AU131579, BE733937, AW404115, AU131993, W86258, BF313032, AA508663, AA311533, AW169592, F17173, AA157549, AW168710, AA854197, BF509014, AW028494, F17657, AA182856, AL516626, AW341596, H21252, AI909957, AA095968, BF025747, AW002276, R29462, AU150634, BE262250, F26847, BG166716, AA156483, AU139963, AU151428, AU126058, AU126364, BG170152, AA773009, BE780783, BE783844, AU137337, AA341400, BF312126, AU153296, BE280416, BE540123, BF315935, X69433, U52144, M86719, X69432, U51167, A74327, A74799, N67429, N90435, and AA115091. HCFBJ91 55 531960 1-871 15-885 AI253987, AI800426, AL042667, AL042670, AI343986, AA515048, BF805088, H63660, BE301610, AU120416, AI697235, AW301950, M78021, AI719298, AI697239, AI697242, BF847612, AW975164, BF769926, AI889995, AW303142, AL036282, AA664126, AW243831, AW468372, AI797998, AI423018, AA744030, AW302081, AI802804, AI620992, AF001298, AC002365, AL135960, AJ131016, AL096840, AL353807, AC007731, AC005500, Z94802, AL022316, AC005231, L78833, AL136223, AC005102, AC005004, AL031311, AL023583, AC004560, AC004033, Z93023, AL031447, AC004471, Z84466, AC005512, AL121891, AC027319, AL136137, AC004650, AC007055, AC005383, AC002565, AC005837, AC009003, AC005548, AC004000, AC021999, AC009086, AL049781, AC005839, AC002425, AC020754, AC007899, AF129756, AC000075, AC011479, Z95115, AL121658, Z93015, AL133324, AL135978, AC009516, AL157877, AC007242, AL096791, AC005355, Z97876, AL031282, AC005484, AC008812, AF217413, AP000211, AP000133, AF207550, AC004799, AL353804, Z92542, AC009314, AL022323, AC009953, AC008848, AC013447, AL109802, AC008733, AC005666, AL049540, AL031075, AC009247, AC007216, AL121586, AC005914, AC005664, AC004087, AL110115, AP000279, AP001718, AL109798, AB015355, AF190464, AL133163, AC002477, AC005522, AC006084, AC002357, AP000512, L78810, AC004846, AL035400, AL049839, AL133507, U91321, AC009756, AC004826, AL049576, AC005103, AC011449, AL121748, AC005722, AL109804, AC002036, AC004448, AF047825, AL132642, AC021016, AF196969, AC004895, AL109806, AC008543, U95742, AC005527, AC002997, AP000500, AP000504, AL138807, AL359382, AC020916, AL008726, AC009477, AC008126, AP000295, AC009194, AC006547, AP000038, AP000106, AC007688, AD000092, AL161670, Z98200, AC006241, AC018720, AL031772, AP000348, AC004106, AC007404, X55448, AK024122, AC005052, AC004167, AC008616, AP000510, AC008623, AC006441, AL031584, AL137162, AC004526, AC008403, AL109935, AC005399, AC005049, AC005585, AC004967, AL353777, AC005229, AL035658, U52112, AL049757, AC002394, Z98941, AC008753, AC006285, AC011464, AC002126, AC012442, AL121890, AL050307, AC002091, AF305872, AL117348, U73634, AC005740, AC004812, AC002504, AL159977, AL022313, AC005020, AC006115, AC004686, AC005015, AL109840, AL035681, AC010422, AC007546, AC004832, AP000113, AP000045, AC000085, AL109743, AL031255, AP001711, AC005529, AC007739, AC005086, AC006023, AC007383, AC018644, AC008395, AL139353, AC007845, AC008745, AF196779, AF165926, AC009228, AC010326, AF215937, AC008392, AC004099, AL034379, AF038458, AC026729, AC007620, AC005094, AC011450, AC011455, AL035458, AC068999, AC007052, AL049539, AC022075, and AC022075. HHFHP90 56 534615 1-1092 15-1106 AW021674, AV759517, AL046620, AI547110, BE139230, AA640305, AV683406, AI254267, AA180056, AI344906, AI318548, AA425283, AA493464, AW839858, AI174703, BE244547, AA524604, AA046906, AW162314, AA280886, AW516250, AI572680, BF724416, AA157876, AI251024, AA631915, AA493245, AW962791, AA019793, AU146789, AI753131, BF837032, AI433952, AW975239, BG180320, AW963552, AW162332, AW020612, AA661583, AW265468, BE042324, AL121039, BE967607, AI702049, AA828840, BF725436, AW631267, BF679568, AA084439, AW473160, AW575808, H62123, BG223498, AI570067, AA804177, AA935827, AA101744, AI114543, AI819419, AI270280, AA994282, AI076729, AA629668, AV763460, AI890297, AW338376, AA171400, BG059139, AA218684, AA604601, AW410844, AI253347, AA313025, AI567676, AI028148, AA167656, AA404619, BE049409, AV755245, AI610814, AA632355, AW514844, AI828721, AI754926, AI457152, BG059924, AW084152, AV760048, AW148821, BE150831, BF681208, R92703, AA133568, AI281622, AA569220, AW029626, AA182928, BF884488, BE045167, AA593168, BG003974, AI469089, AW850985, AA362439, BF882222, AI744963, BF846619, AI888050, AU157188, AW963295, AI376687, AW962971, AV728973, AI921744, AV764119, AA578711, AA600127, AI884404, BF207643, AA664963, BE152006, AI434103, AA622637, AI520984, AW880986, AA084320, BF914416, BF790866, BF849260, AA298365, AI003068, BF739035, BG250794, AI064968, W03800, AW243817, AA515727, AI308529, AW974283, BF944618, AI889177, AU158814, BF904892, AI745666, BF882223, AA779599, N49298, AI744890, BE044000, AU120977, AI275631, AA554289, AW157128, AW771679, BF942991, AI860648, BF970107, AA315052, AA601336, BF880881, AA491941, AI307563, AA047057, N99245, AA746951, AW028376, AA776663, AA729004, BE156611, AW022796, AA018258, BE875478, N26159, AA179364, AA244267, AI890857, AI907506, AI274773, BE155101, AA618531, AA487053, H47461, AI300608, AC009032, AC004938, AL135818, AP001670, AC005839, AF200465, AC008518, AC002326, AC011462, AC004223, AL009031, AC005914, AL022318, AC002524, AC022316, AF205588, AC007686, AK024511, AP000553, AL096701, AL034423, AL035587, AC000353, AC007919, AL356747, AC005015, AC007051, AE111169, AP001052, AL022323, AC018738, AP001752, AC005377, AC006252, AC005516, AC011450, AP000963, AC008543, AC011529, AL135749, AP001716, AC005368, AC005911, AC008760, Z85987, AC007021, AC006942, U80017, AL117337, AL133332, AP000289, AC010877, AP000042, AP000110, AC016554, AC006120, AC004955, AL121580, AL136223, Z97056, AC006440, AL353804, L47222, AL022320, AL109797, AL133448, AC011480, AL031663, AC008403, AC009314, AC007722, AC006511, AC006211, AC002350, AC016995, AC006277, AF023268, AC008536, AC004999, AP000502, AP000365, AC007956, U89337, AP000547, AJ400877, AC005412, Z68756, AL157760, AL049547, AC011510, AL121586, AL136967, AL050331, AL365444, AC011559, AC010326, AC005301, AL031662, AC003074, AC003029, AC005581, AC008764, AC005000, AC003665, AC003037, AK026694, U52852, AC005484, AC005730, AL133163, AF168787, AC010527, AL031846, AL031680, AC010363, AC009003, AL049569, Z82244, AC007345, AL133355, AC006261, AL050349, AL020993, AC008264, AL157952, AC005066, AL096814, AC007878, AL008726, AD000864, AF067844, AC008812, AC007263, Z73979, AC011475, AC020626, AC002553, AC009756, AC009060, AC002477, AL035460, AC008070, AC005779, AC004006, AL353807, AL136992, AK026729, AC006312, AC009530, AC002126, AL033529, AC012309, AL109758, AL391995, AL357497, AP000031, AC005335, AL353653, AC005821, AL136228, AL033533, AP000039, U50871, AL136418, AC006204, AL139182, AL109935, AF317635, AL020997, AC005755, AL161937, AP000280, AL136295, AL133211, Z93931, AL133418, AF190464, Z86090, AC007240, AC005740, AC005049, AL136362, AL024498, AL022315, AL031281, AC009477, AP001727, AC002303, AL139100, AC004231, AC005618, AB035195, AC004783, AC007098, AP000107, AC004847, AP000343, Z93017, AC005084, AC006064, Z82184, AL031229, AC006449, AC004967, AC009455, AC006974, AC008651, AF165926, AL122001, AC009399, AL355476, AC021016, AC010480, AF228703, AC005840, AL031311, AJ003147, AC006969, AL050335, Z83840, AC002464, AC019155, AC006345, AC005358, AL136971, AC020552, AC006487, AL354696, AL022165, AP000337, AL035072, AC005726, AL078639, AX026165, AC005207, AC017092, AC004814, AP000694, AC011490, AC000075, AL031295, AL121601, AP000215, Z93020, AC002544, AC007216, Z81008, AC010328, AC073481, AP001362, AP001362, AC022488, AC022488, AC022488, AP000803, AP000803, AP001459, and AP001459. HLYCQ48 57 527757 1-750 15-764 BE139139, AI250552, AI254770, AI284543, AI821273, AI251284, AI251034, AI251203, AI223626, BE138387, AW970571, AI249853, AW238341, AW303098, AI791718, BE042006, AA501867, AW890425, AA613630, BF805088, AA676592, AA682912, AW970987, AW276678, BF916055, AI973173, AA574000, H68343, BE139133, AV743776, BE676912, AL037929, AW302017, AW162288, AW270771, AL037932, AL037923, AV743864, AA873573, AA302661, AA572983, AW338506, AI343986, AW236219, AV762633, AA525293, AA503018, AL042667, AL042670, AW591754, BF764256, C06141, BF870951, AI431434, AB039887, AP001628, AP001748, AC007227, AL135927, AC005738, AL358777, AL021154, U78308, AC002310, AC010458, AC006483, AL022319, AF141309, AC005339, AL034420, AC025588, I34294, AC006511, AF031078, AC005261, AL109627, AC005840, AP001727, U95742, AL022322, AL137129, AC002316, AC007226, AC003109, AP000346, AL121900, Z83844, AP001753, AC024078, AC007057, AL049872, AL121952, AL035704, AC022515, AP000501, AC008760, AL035400, AF196969, AF030876, AC006487, AL121897, AL049829, AC018633, AC004491, AC005932, AC005863, AC009060, AL022320, AC006312, AL031602, AC008745, AL122015, AC006512, Z98304, AC011464, AL354864, AC004033, AL035464, AC011500, AC007249, AC005251, AF036581, AC007216, AC020916, AF196779, AC005920, AC005924, AL020995, AP000347, AC002375, AC004832, AC020947, Z92546, AC005081, AC005488, AC005837, AC005088, AC007653, AC007687, AC011290, AL049709, AC008821, AC008924, AL022311, AC007850, AL355916, AC007404, AF064090, AC006041, AF045555, U91326, AC006251, AC000025, AL033529, AC009228, AC008115, AL136304, AL024498, AL137073, AC005156, AC007845, AL034405, AC005527, AL353804, AC008543, AC008764, AC008403, AC007679, AL031672, AL121586, AF243527, AL121751, AF317635, AC004149, AL139331, AP000275, AC004953, AC018751, AL031007, AP000353, AL137918, AL109928, U47924, AL133174, AC016026, AL353653, AP000022, AC002544, AC004645, AP000553, AL008719, AL121899, AC011497, AC016025, AC009516, AC011479, AL109984, Z83733, AC002553, AP000105, AP000037, AC008392, AF038458, AC011551, AP000359, AC005102, AL158040, AC004883, AL022336, AC007051, AL161725, AC006241, AL139100, AP001426, AC008747, AL135744, Z83845, AL139392, AC011449, AC009086, AC007565, AL390026, Z82215, AC004025, AC007191, Z98749, AC005049, AC006257, AC005529, AL031280, AL022165, AC003030, AL049749, AB041992, AP000114, AP000046, AC004089, AP001752, AP001725, AC005071, AL138787, AC009517, AC007405, AL109921, AC005618, AC020898, AC008537, AL035683, AP000696, AC007731, AL034379, U07563, AC003041, AL358372, AL020993, AC008569, AL031848, AL022476, AC002559, AL157406, AC027319, AP001712, AC006111, AC011462, AC004222, AC005778, AL132777, AC005593, AL117381, AC005274, AL031284, AC021036, AL022163, AJ400877, AF207550, AL157838, and AP000350. HHLAB07 58 532602 1-724 15-738 AA309074, and AC010764. HFOXE30 59 532120 1-427 15-441 AF327179, AL031273, Z96270, AL031273, AL031273, AL031273, and AC074234. HBJEL68 60 531924 1-770 15-784 BG150876, AI741650, AL159973, and AL159973. HFOXA73 61 850699 1-526 15-540 AC005866, AC007618, and AC005866. HFIUR35 62 532062 1-590 15-604 AA127598, T77777, AC009778, AC063924, and AC063924. HFPDE86 63 527728 1-738 15-752 AA649642, AI963720, AA610491, AI431303, BF668217, AA507824, AI224093, AW504669, AI580652, BE742023, AV759204, BE160516, AW023672, AA226153, AA664581, AA806796, AW979060, AA601492, BF680041, AA523837, AA613227, BE677026, AV764329, AI589230, AL042420, AA503015, AW169537, AV691147, F25867, AV760543, AC007390, AC009060, AP000557, AC005531, AC016026, AL353807, AC011495, AC005229, AL354720, AC007957, AC011442, AL009181, AF001552, AF088219, AL356414, AC006483, AP000201, AC005081, AC009756, AP000097, AC010271, AC009244, AC007956, AP000240, AC004675, AP000500, AC007216, AC008760, AL121655, U95742, AC008764, AC023490, AL353748, Z93017, AC007226, AC003962, AL133448, AP000556, AL161670, AL109825, AC008569, AC005004, AP001705, AL034417, AC004812, AL033392, AC005258, AC010513, AC007676, Z83840, AC010289, AC018682, AC004166, AC004971, AC007999, AL023494, Z98744, AL050348, AC004965, AL352984, AC006011, AC010168, AC004895, AL117336, AL136137, AC005071, AC007055, AF243527, AL096712, AL133289, AL022318, AC005520, AP001331, AC006207, AP001708, AL023575, AC005015, AC006241, AL023879, AL136179, AC004089, AC027319, AC008518, AL109804, AC009228, AL359457, AP001748, AC016637, AL138787, AC003070, AP001629, AC007227, AC008687, U91322, AL137073, AP000140, AC002303, AC005730, AJ277557, AP001692, AC008753, AC010582, AC005776, AC009247, AL009179, AC020750, AC010150, AL157838, AC007619, AL132639, AL135927, U89336, AL121983, AC016627, AL157938, AF165926, AC005231, AC007537, AC007050, AL139023, AL049780, AL442166, AP000088, AC005011, AL031904, AC004887, Z93930, AC003109, AL163284, AJ009614, AC002425, AC009470, AP001759, AC009086, AL117186, AP001753, AC004922, Z98050, AC005355, AL034420, AC006571, AC006079, AL080242, AL163301, AC007390, AC007390, and AC007390.

[0467] TABLE 4 Code Description Tissue Organ Cell Line Disease Vector AR022 a_Heart a_Heart AR023 a_Liver a_Liver AR024 a_mammary gland a_mammary gland AR025 a_Prostate a_Prostate AR026 a_small intestine a_small intestine AR027 a_Stomach a_Stomach AR028 Blood B cells Blood B cells AR029 Blood B cells activated Blood B cells activated AR030 Blood B cells resting Blood B cells resting AR031 Blood T cells activated Blood T cells activated AR032 Blood T cells resting Blood T cells resting AR033 brain brain AR034 breast breast AR035 breast cancer breast cancer AR036 Cell Line CAOV3 Cell Line CAOV3 AR037 cell line PA-1 cell line PA-1 AR038 cell line transformed cell line transformed AR039 colon colon AR040 colon (9808co65R) colon (9808co65R) AR041 colon (9809co15) colon (9809co15) AR042 colon cancer colon cancer AR043 colon cancer colon cancer (9808co64R) (9808co64R) AR044 colon cancer 9809co14 colon cancer 9809co14 AR045 corn clone 5 corn clone 5 AR046 corn clone 6 corn clone 6 AR047 corn clone 2 corn clone 2 AR048 corn clone 3 corn clone 3 AR049 Corn Clone 4 Corn Clone 4 AR050 Donor II B Cells 24 hrs Donor II B Cells 24 hrs AR051 Donor II B Cells 72 hrs Donor II B Cells 72 hrs AR052 Donor II B-Cells 24 hrs. Donor II B-Cells 24 hrs. AR053 Donor II B-Cells 72 hrs Donor II B-Cells 72 hrs AR054 Donor II Resting B Cells Donor II Resting B Cells AR055 Heart Heart AR056 Human Lung (clonetech) Human Lung (clonetech) AR057 Human Mammary Human Mammary (clontech) (clontech) AR058 Human Thymus Human Thymus (clonetech) (clonetech) AR059 Jurkat (unstimulated) Jurkat (unstimulated) AR060 Kidney Kidney AR061 Liver Liver AR062 Liver (Clontech) Liver (Clontech) AR063 Lymphocytes chronic Lymphocytes chronic lymphocytic leukaemia lymphocytic leukaemia AR064 Lymphocytes diffuse Lymphocytes diffuse large B cell lymphoma large B cell lymphoma AR065 Lymphocytes follicular Lymphocytes lymphoma follicular lymphoma AR066 normal breast normal breast AR067 Normal Ovarian Normal Ovarian (4004901) (4004901) AR068 Normal Ovary Normal Ovary 9508G045 9508G045 AR069 Normal Ovary Normal Ovary 9701G208 9701G208 AR070 Normal Ovary Normal Ovary 9806G005 9806G005 AR071 Ovarian Cancer Ovarian Cancer AR072 Ovarian Cancer Ovarian Cancer (9702G001) (9702G001) AR073 Ovarian Cancer Ovarian Cancer (9707G029) (9707G029) AR074 Ovarian Cancer Ovarian Cancer (9804G011) (9804G011) AR075 Ovarian Cancer Ovarian Cancer (9806G019) (9806G019) AR076 Ovarian Cancer Ovarian Cancer (9807G017) (9807G017) AR077 Ovarian Cancer Ovarian Cancer (9809G001) (9809G001) AR078 ovarian cancer 15799 ovarian cancer 15799 AR079 Ovarian Cancer Ovarian Cancer 17717AID 17717AID AR080 Ovarian Cancer Ovarian Cancer 4004664B1 4004664B1 AR081 Ovarian Cancer Ovarian Cancer 4005315A1 4005315A1 AR082 ovarian cancer ovarian cancer 94127303 94127303 AR083 Ovarian Cancer Ovarian Cancer 96069304 96069304 AR084 Ovarian Cancer Ovarian Cancer 9707G029 9707G029 AR085 Ovarian Cancer Ovarian Cancer 9807G045 9807G045 AR086 ovarian cancer ovarian cancer 9809G001 9809G001 AR087 Ovarian Cancer Ovarian Cancer 9905C032RC 9905C032RC AR088 Ovarian cancer 9907 Ovarian cancer 9907 C00 3rd C00 3rd AR089 Prostate Prostate AR090 Prostate (clonetech) Prostate (clonetech) AR091 prostate cancer prostate cancer AR092 prostate cancer #15176 prostate cancer #15176 AR093 prostate cancer #15509 prostate cancer #15509 AR094 prostate cancer #15673 prostate cancer #15673 AR095 Small Intestine Small Intestine (Clontech) (Clontech) AR096 Spleen Spleen AR097 Thymus T cells activated Thymus T cells activated AR098 Thymus T cells resting Thymus T cells resting AR099 Tonsil Tonsil AR100 Tonsil geminal center Tonsil geminal center centroblast centroblast AR101 Tonsil germinal center B Tonsil germinal cell center B cell AR102 Tonsil lymph node Tonsil lymph node AR103 Tonsil memory B cell Tonsil memory B cell AR104 Whole Brain Whole Brain AR105 Xenograft ES-2 Xenograft ES-2 AR106 Xenograft SW626 Xenograft SW626 AR119 001: IL-2 001: IL-2 AR120 001: IL-2.1 001: IL-2.1 AR121 001: IL-2_b 001: IL-2_b AR124 002: Monocytes 002: Monocytes untreated (1 hr) untreated (1 hr) AR125 002: Monocytes 002: Monocytes untreated (5 hrs) untreated (5 hrs) AR126 002: Control.1C 002: Control.1C AR127 002: 1L2.1C 002: 1L2.1C AR130 003: Placebo-treated 003: Placebo-treated Rat Lacrimal Gland Rat Lacrimal Gland AR131 003: Placebo-treated 003: Placebo-treated Rat Submandibular Rat Submandibular Gland Gland AR135 004: Monocytes 004: Monocytes untreated (5 hrs) untreated (5 hrs) AR136 004: Monocytes 004: Monocytes untreated 1 hr untreated 1 hr AR139 005: Placebo (48 hrs) 005: Placebo (48 hrs) AR140 006: pC4 (24 hrs) 006: pC4 (24 hrs) AR141 006: pC4 (48 hrs) 006: pC4 (48 hrs) AR152 007: PHA (1 hr) 007: PHA (1 hr) AR153 007: PHA (6 HRS) 007: PHA (6 HRS) AR154 007: PMA (6 hrs) 007: PMA (6 hrs) AR155 008: 1449_#2 008: 1449_#2 AR161 01: A - max 24 01: A - max 24 AR162 01: A - max 26 01: A - max 26 AR163 01: A - max 30 01: A - max 30 AR164 01: B - max 24 01: B - max 24 AR165 01: B - max 26 01: B - max 26 AR166 01: B - max 30 01: B - max 30 AR167 1449 Sample 1449 Sample AR168 3T3P10 1.0 uM insulin 3T3P10 1.0 uM insulin AR169 3T3P10 10 nM Insulin 3T3P10 10 nM Insulin AR170 3T3P10 10 uM insulin 3T3P10 10 uM insulin AR171 3T3P10 No Insulin 3T3P10 No Insulin AR172 3T3P4 3T3P4 AR173 Adipose (41892) Adipose (41892) AR174 Adipose Diabetic Adipose Diabetic (41611) (41611) AR175 Adipose Diabetic Adipose Diabetic (41661) (41661) AR176 Adipose Diabetic Adipose Diabetic (41689) (41689) AR177 Adipose Diabetic Adipose Diabetic (41706) (41706) AR178 Adipose Diabetic Adipose Diabetic (42352) (42352) AR179 Adipose Diabetic Adipose Diabetic (42366) (42366) AR180 Adipose Diabetic Adipose Diabetic (42452) (42452) AR181 Adipose Diabetic Adipose Diabetic (42491) (42491) AR182 Adipose Normal (41843) Adipose Normal (41843) AR183 Adipose Normal (41893) Adipose Normal (41893) AR184 Adipose Normal (42452) Adipose Normal (42452) AR185 Adrenal Gland Adrenal Gland AR186 Adrenal Gland + Whole Adrenal Gland + Brain Whole Brain AR187 B7 (1 hr) + (inverted) B7 (1 hr) + (inverted) AR188 Breast (18275A2B) Breast (18275A2B) AR189 Breast (4004199) Breast (4004199) AR190 Breast (4004399) Breast (4004399) AR191 Breast (4004943B7) Breast (4004943B7) AR192 Breast (4005570B1) Breast (4005570B1) AR193 Breast Cancer Breast Cancer (4004127A30) (4004127A30) AR194 Breast Cancer Breast Cancer (400443A21) (400443A21) AR195 Breast Cancer Breast Cancer (4004643A2) (4004643A2) AR196 Breast Cancer Breast Cancer (4004710A7) (4004710A7) AR197 Breast Cancer Breast Cancer (4004943A21) (4004943A21) AR198 Breast Cancer Breast Cancer (400553A2) (400553A2) AR199 Breast Cancer Breast Cancer (9805C046R) (9805C046R) AR200 Breast Cancer Breast Cancer (9806C012R) (9806C012R) AR201 Breast Cancer (ODQ Breast Cancer (ODQ 45913) 45913) AR202 Breast Cancer Breast Cancer (ODQ45913) (ODQ45913) AR203 Breast Cancer Breast Cancer (ODQ4591B) (ODQ4591B) AR204 Colon Cancer (15663) Colon Cancer (15663) AR205 Colon Cancer Colon Cancer (4005144A4) (4005144A4) AR206 Colon Cancer Colon Cancer (4005413A4) (4005413A4) AR207 Colon Cancer Colon Cancer (4005570B1) (4005570B1) AR208 Control RNA #1 Control RNA #1 AR209 Control RNA #2 Control RNA #2 AR210 Cultured Preadipocyte Cultured (blue) Preadipocyte (blue) AR211 Cultured Preadipocyte Cultured (Red) Preadipocyte (Red) AR212 Donor II B-Cells 24 hrs Donor II B-Cells 24 hrs AR213 Donor II Resting B-Cells Donor II Resting B- Cells AR214 H114EP12 10 nM H114EP12 10 nM Insulin Insulin AR215 H114EP12 (10 nM H114EP12 (10 nM insulin) insulin) AR216 H114EP12 (2.6 ug/ul) H114EP12 (2.6 ug/ul) AR217 H114EP12 (3.6 ug/ul) H114EP12 (3.6 ug/ul) AR218 HUVEC #1 HUVEC #1 AR219 HUVEC #2 HUVEC #2 AR221 L6 undiff. L6 undiff. AR222 L6 Undifferentiated L6 Undifferentiated AR223 L6P8 + 10 nM Insulin L6P8 + 10 nM Insulin AR224 L6P8 + HS L6P8 + HS AR225 L6P8 10 nM Insulin L6P8 10 nM Insulin AR226 Liver (00-06-A007B) Liver (00-06-A007B) AR227 Liver (96-02-A075) Liver (96-02-A075) AR228 Liver (96-03-A144) Liver (96-03-A144) AR229 Liver (96-04-A138) Liver (96-04-A138) AR230 Liver (97-10-A074B) Liver (97-10-A074B) AR231 Liver (98-09-A242A) Liver (98-09-A242A) AR232 Liver Diabetic (1042) Liver Diabetic (1042) AR233 Liver Diabetic (41616) Liver Diabetic (41616) AR234 Liver Diabetic (41955) Liver Diabetic (41955) AR235 Liver Diabetic (42352R) Liver Diabetic (42352R) AR236 Liver Diabetic (42366) Liver Diabetic (42366) AR237 Liver Diabetic (42483) Liver Diabetic (42483) AR238 Liver Diabetic (42491) Liver Diabetic (42491) AR239 Liver Diabetic (99-09- Liver Diabetic (99- A281A) 09-A281A) AR240 Lung Lung AR241 Lung (27270) Lung (27270) AR242 Lung (2727Q) Lung (2727Q) AR243 Lung Cancer Lung Cancer (4005116A1) (4005116A1) AR244 Lung Cancer Lung Cancer (4005121A5) (4005121A5) AR245 Lung Cancer Lung Cancer (4005121A5)) (4005121A5)) AR246 Lung Cancer Lung Cancer (4005340A4) (4005340A4) AR247 Mammary Gland Mammary Gland AR248 Monocyte (CT) Monocyte (CT) AR249 Monocyte (OCT) Monocyte (OCT) AR250 Monocytes (CT) Monocytes (CT) AR251 Monocytes (INFG 18 hr) Monocytes (INFG 18 hr) AR252 Monocytes (INFG 18 hr) Monocytes (INFG 18 hr) AR253 Monocytes (INFG 8-11) Monocytes (INFG 8-11) AR254 Monocytes (O CT) Monocytes (O CT) AR255 Muscle (91-01-A105) Muscle (91-01-A105) AR256 Muscle (92-04-A059) Muscle (92-04-A059) AR257 Muscle (97-11-A056d) Muscle (97-11- A056d) AR258 Muscle (99-06-A210A) Muscle (99-06- A210A) AR259 Muscle (99-07-A203B) Muscle (99-07- A203B) AR260 Muscle (99-7-A203B) Muscle (99-7- A203B) AR261 Muscle Diabetic Muscle Diabetic (42352R) (42352R) AR262 Muscle Diabetic (42366) Muscle Diabetic (42366) AR263 NK-19 Control NK-19 Control AR264 NK-19 IL Treated 72 hrs NK-19 IL Treated 72 hrs AR265 NK-19 UK Treated 72 hrs. NK-19 UK Treated 72 hrs. AR266 Omentum Normal (94- Omentum Normal 08-B009) (94-08-B009) AR267 Omentum Normal (97- Omentum Normal 01-A039A) (97-01-A039A) AR268 Omentum Normal (97- Omentum Normal 04-A114C) (97-04-A114C) AR269 Omentum Normal (97- Omentum Normal 06-A117C) (97-06-A117C) AR270 Omentum Normal (97- Omentum Normal 09-B004C) (97-09-B004C) AR271 Ovarian Cancer Ovarian Cancer (17717AID) (17717AID) AR272 Ovarian Cancer Ovarian Cancer (9905C023RC) (9905C023RC) AR273 Ovarian Cancer Ovarian Cancer (9905C032RC) (9905C032RC) AR274 Ovary (9508G045) Ovary (9508G045) AR275 Ovary (9701G208) Ovary (9701G208) AR276 Ovary 9806G005 Ovary 9806G005 AR277 Pancreas Pancreas AR278 Placebo Placebo AR279 rIL2 Control rIL2 Control AR280 RSS288L RSS288L AR281 RSS288LC RSS288LC AR282 Salivary Gland Salivary Gland AR283 Skeletal Muscle Skeletal Muscle AR284 Skeletal Muscle (91-01- Skeletal Muscle (91- A105) 01-A105) AR285 Skeletal Muscle (42180) Skeletal Muscle (42180) AR286 Skeletal Muscle (42386) Skeletal Muscle (42386) AR287 Skeletal Muscle (42461) Skeletal Muscle (42461) AR288 Skeletal Muscle (91-01- Skeletal Muscle (91- A105) 01-A105) AR289 Skeletal Muscle (92-04- Skeletal Muscle (92- A059) 04-A059) AR290 Skeletal Muscle (96-08- Skeletal Muscle (96- A171) 08-A171) AR291 Skeletal Muscle (97-07- Skeletal Muscle (97- A190A) 07-A190A) AR292 Skeletal Muscle Diabetic Skeletal Muscle (42352) Diabetic (42352) AR293 Skeletal Muscle Diabetic Skeletal Muscle (42366) Diabetic (42366) AR294 Skeletal Muscle Diabetic Skeletal Muscle (42395) Diabetic (42395) AR295 Skeletal Muscle Diabetic Skeletal Muscle (42483) Diabetic (42483) AR296 Skeletal Muscle Diabetic Skeletal Muscle (42491) Diabetic (42491) AR297 Skeletal Muscle Diabetic Skeletal Muscle 42352 Diabetic 42352 AR298 Skeletal Musle (42461) Skeletal Musle (42461) AR299 Small Intestine Small Intestine AR300 Stomach Stomach AR301 T-Cell + HDPBQ71.fc T-Cell + 1449 16 hrs HDPBQ71.fc 1449 16 hrs AR302 T-Cell + HDPBQ71.fc T-Cell + 1449 6 hrs HDPBQ71.fc 1449 6 hrs AR303 T-Cell + IL2 16 hrs T-Cell + IL2 16 hrs AR304 T-Cell + IL2 6 hrs T-Cell + IL2 6 hrs AR306 T-Cell Untreated 16 hrs T-Cell Untreated 16 hrs AR307 T-Cell Untreated 6 hrs T-Cell Untreated 6 hrs AR308 T-Cells 24 hours T-Cells 24 hours AR309 T-Cells 24 hrs T-Cells 24 hrs AR310 T-Cells 24 hrs. T-Cells 24 hrs. AR311 T-Cells 24 hrs T-Cells 24 hrs AR312 T-Cells 4 days T-Cells 4 days AR313 Thymus Thymus AR314 TRE TRE AR315 TREC TREC AR316 Virtual Mixture Virtual Mixture H0009 Human Fetal Brain Uni-ZAP XR H0012 Human Fetal Kidney Human Fetal Kidney Kidney Uni-ZAP XR H0013 Human 8 Week Whole Human 8 Week Old Embryo Uni-ZAP XR Embryo Embryo H0018 Human Greater Human Greater peritoneum Uni-ZAP XR Omentum, fII remake Omentum H0024 Human Fetal Lung III Human Fetal Lung Lung Uni-ZAP XR H0026 Namalwa Cells Namalwa B-Cell Lambda ZAP Line, EBV II immortalized H0031 Human Placenta Human Placenta Placenta Uni-ZAP XR H0032 Human Prostate Human Prostate Prostate Uni-ZAP XR H0036 Human Adult Small Human Adult Small Small Int. Uni-ZAP XR Intestine Intestine H0038 Human Testes Human Testes Testis Uni-ZAP XR H0041 Human Fetal Bone Human Fetal Bone Bone Uni-ZAP XR H0046 Human Endometrial Human Endometrial Uterus disease Uni-ZAP XR Tumor Tumor H0048 Human Pineal Gland Human Pineal Gland Uni-ZAP XR H0050 Human Fetal Heart Human Fetal Heart Heart Uni-ZAP XR H0051 Human Hippocampus Human Hippocampus Brain Uni-ZAP XR H0052 Human Cerebellum Human Cerebellum Brain Uni-ZAP XR H0081 Human Fetal Epithelium Human Fetal Skin Skin Uni-ZAP XR (Skin) H0083 HUMAN JURKAT Jurkat Cells Uni-ZAP XR MEMBRANE BOUND POLYSOMES H0087 Human Thymus Human Thymus pBluescript H0090 Human T-Cell T-Cell Lymphoma T-Cell disease Uni-ZAP XR Lymphoma H0095 Human Greater Human Greater peritoneum Uni-ZAP XR Omentum, RNA Omentum Remake H0099 Human Lung Cancer, Human Lung Cancer Lung pBluescript subtracted H0100 Human Whole Six Week Human Whole Six Embryo Uni-ZAP XR Old Embryo Week Old Embryo H0111 Human Placenta, Human Placenta Placenta pBluescript subtracted H0123 Human Fetal Dura Mater Human Fetal Dura Brain Uni-ZAP XR Mater H0124 Human Human Sk Muscle disease Uni-ZAP XR Rhabdomyosarcoma Rhabdomyosarcoma H0134 Raji Cells, Cyclohexamide Blood Cell Line Uni-ZAP XR cyclohexamide treated Treated Cem, Jurkat, Raji, and Supt H0135 Human Synovial Human Synovial Synovium Uni-ZAP XR Sarcoma Sarcoma H0136 Supt Cells, Cyclohexamide Blood Cell Line Uni-ZAP XR cyclohexamide treated Treated Cem, Jurkat, Raji, and Supt H0144 Nine Week Old Early 9 Wk Old Early Embryo Uni-ZAP XR Stage Human Stage Human H0149 7 Week Old Early Stage Human Whole 7 Embryo Uni-ZAP XR Human, subtracted Week Old Embryo H0154 Human Fibrosarcoma Human Skin Skin disease Uni-ZAP XR Fibrosarcoma H0156 Human Adrenal Gland Human Adrenal Adrenal disease Uni-ZAP XR Tumor Gland Tumor Gland H0163 Human Synovium Human Synovium Synovium Uni-ZAP XR H0165 Human Prostate Cancer, Human Prostate Prostate disease Uni-ZAP XR Stage B2 Cancer, stage B2 H0169 Human Prostate Cancer, Human Prostate Prostate disease Uni-ZAP XR Stage C fraction Cancer, stage C H0170 12 Week Old Early Twelve Week Old Embryo Uni-ZAP XR Stage Human Early Stage Human H0171 12 Week Old Early Twelve Week Old Embryo Uni-ZAP XR Stage Human, II Early Stage Human H0172 Human Fetal Brain, Human Fetal Brain Brain Lambda ZAP random primed II H0178 Human Fetal Brain Human Fetal Brain Brain Uni-ZAP XR H0181 Human Primary Breast Human Primary Breast disease Uni-ZAP XR Cancer Breast Cancer H0196 Human Human Heart Uni-ZAP XR Cardiomyopathy, Cardiomyopathy subtracted H0199 Human Fetal Liver, Human Fetal Liver Liver Uni-ZAP XR subtracted, neg clone H0208 Early Stage Human Human Fetal Lung Lung pBluescript Lung, subtracted H0211 Human Human Prostate Prostate pBluescript Prostate, differential expression H0213 Human Pituitary, Human Pituitary Uni-ZAP XR subtracted H0216 Supt cells, Cyclohexamide Blood Cell Line pBluescript cyclohexamide treated, Treated Cem, Jurkat, subtracted Raji, and Supt H0228 C7MCF7 cell line, C7MCF7 Cell Line, Breast Cell Line Uni-ZAP XR estrogen treated estrogen treated H0239 Human Kidney Tumor Human Kidney Kidney disease Uni-ZAP XR Tumor H0254 Breast Lymph node Breast Lymph Node Lymph Uni-ZAP XR cDNA library Node H0255 breast lymph node Breast Lymph Node Lymph Lambda ZAP CDNA library Node II H0264 human tonsils Human Tonsil Tonsil Uni-ZAP XR H0265 Activated T-Cell T-Cells Blood Cell Line Uni-ZAP XR (12 hs)/Thiouridine labelledEco H0266 Human Microvascular HMEC Vein Cell Line Lambda ZAP Endothelial Cells, fract. A II H0271 Human Neutrophil, Human Neutrophil - Blood Cell Line Uni-ZAP XR Activated Activated H0272 HUMAN TONSILS, Human Tonsil Tonsil Uni-ZAP XR FRACTION 2 H0288 Human OB HOS control Human Bone Cell Line Uni-ZAP XR fraction I Osteoblastoma HOS cell line H0294 Amniotic Cells - TNF Amniotic Cells - Placenta Cell Line Uni-ZAP XR induced TNF induced H0295 Amniotic Cells - Amniotic Cells - Placenta Cell Line Uni-ZAP XR Primary Culture Primary Culture H0305 CD34 positive cells CD34 Positive Cells Cord ZAP Express (Cord Blood) Blood H0306 CD34 depleted Buffy CD34 Depleted Cord ZAP Express Coat (Cord Blood) Buffy Coat (Cord Blood Blood) H0309 Human Chronic Synovium, Chronic Synovium disease Uni-ZAP XR Synovitis Synovitis/ Osteoarthritis H0316 HUMAN STOMACH Human Stomach Stomach Uni-ZAP XR H0318 HUMAN B CELL Human B Cell Lymph disease Uni-ZAP XR LYMPHOMA Lymphoma Node H0328 human ovarian cancer Ovarian Cancer Ovary disease Uni-ZAP XR H0333 Hemangiopericytoma Hemangiopericytoma Blood disease Lambda ZAP vessel II H0341 Bone Marrow Cell Line Bone Marrow Cell Bone Cell Line Uni-ZAP XR (RS4; 11) Line RS4; 11 Marrow H0351 Glioblastoma Glioblastoma Brain disease Uni-ZAP XR H0355 Human Liver Human Liver, normal pCMVSport 1 Adult H0370 H. Lymph node breast Lymph node with disease Uni-ZAP XR Cancer Met. Breast Cancer H0373 Human Heart Human Adult Heart Heart pCMVSport 1 H0393 Fetal Liver, subtraction Human Fetal Liver Liver pBluescript II H0402 CD34 depleted Buffy CD34 Depleted Cord ZAP Express Coat (Cord Blood), re- Buffy Coat (Cord Blood excision Blood) H0422 T-Cell PHA 16 hrs T-Cells Blood Cell Line pSport1 H0423 T-Cell PHA 24 hrs T-Cells Blood Cell Line pSport1 H0424 Human Pituitary, subt Human Pituitary pBluescript IX H0427 Human Adipose Human Adipose, left pSport1 hiplipoma H0431 H. Kidney Medulla, re- Kidney medulla Kidney pBluescript excision H0435 Ovarian Tumor 10-3-95 Ovarian Tumor, Ovary pCMVSport OV350721 2.0 H0436 Resting T-Cell Library, II T-Cells Blood Cell Line pSport1 H0441 H. Kidney Cortex, Kidney cortex Kidney pBluescript subtracted H0445 Spleen, Chronic Human Spleen, CLL Spleen disease pSport1 lymphocytic leukemia H0478 Salivary Gland, Lib 2 Human Salivary Salivary pSport1 Gland gland H0483 Breast Cancer cell line, Breast Cancer Cell pSport1 MDA 36 line, MDA 36 H0484 Breast Cancer Cell line, Breast Cancer Cell pSport1 angiogenic line, Angiogenic, 36T3 H0486 Hodgkin's Lymphoma II Hodgkin's disease pCMVSport Lymphoma II 2.0 H0494 Keratinocyte Keratinocyte pCMVSport 2.0 H0497 HEL cell line HEL cell line HEL pSport1 92.1.7 H0506 Ulcerative Colitis Colon Colon pSport1 H0509 Liver, Hepatoma Human Liver, Liver disease pCMVSport Hepatoma, patient 8 3.0 H0510 Human Liver, normal Human Liver, Liver pCMVSport normal, Patient # 8 3.0 H0518 pBMC stimulated w/ pBMC stimulated pCMVSport poly I/C with poly I/C 3.0 H0520 NTERA2 + retinoic NTERA2, pSport1 acid, 14 days Teratocarcinoma cell line H0521 Primary Dendritic Cells, Primary Dendritic pCMVSport lib 1 cells 3.0 H0522 Primary Dendritic Primary Dendritic pCMVSport cells, frac 2 cells 3.0 H0529 Myoloid Progenitor Cell TF-1 Cell Line; pCMVSport Line Myoloid progenitor 3.0 cell line H0530 Human Dermal Human Dermal pSport1 Endothelial Endothelial Cells; Cells, untreated untreated H0542 T Cell helper I Helper T cell pCMVSport 3.0 H0543 T cell helper II Helper T cell pCMVSport 3.0 H0544 Human endometrial Human endometrial pCMVSport stromal cells stromal cells 3.0 H0545 Human endometrial Human endometrial pCMVSport stromal cells-treated stromal cells-treated 3.0 with progesterone with proge H0547 NTERA2 NTERA2, pSport1 teratocarcinoma cell Teratocarcinoma cell line + retinoic acid (14 line days) H0549 H. Epididiymus, caput & Human Epididiymus, Uni-ZAP XR corpus caput and corpus H0550 H. Epididiymus, cauda Human Epididiymus, Uni-ZAP XR cauda H0551 Human Thymus Stromal Human Thymus pCMVSport Cells Stromal Cells 3.0 H0553 Human Placenta Human Placenta pCMVSport 3.0 H0555 Rejected Kidney, lib 4 Human Rejected Kidney disease pCMVSport Kidney 3.0 H0556 Activated T- T-Cells Blood Cell Line Uni-ZAP XR cell(12 h)/Thiouridine-re- excision H0560 KMH2 KMH2 pCMVSport 3.0 H0561 L428 L428 pCMVSport 3.0 H0562 Human Fetal Brain, Human Fetal Brain pCMVSport normalized c5-11-26 2.0 H0571 Human Fetal Brain, Human Fetal Brain pCMVSport normalized C500HE 2.0 H0574 Hepatocellular Tumor; Hepatocellular Liver disease Lambda ZAP re-excision Tumor II H0575 Human Adult Human Adult Lung Uni-ZAP XR Pulmonary; re-excision Pulmonary H0580 Dendritic cells, pooled Pooled dendritic cells pCMVSport 3.0 H0581 Human Bone Marrow, Human Bone Marrow Bone pCMVSport treated Marrow 3.0 H0586 Healing groin wound, healing groin wound, groin disease pCMVSport 6.5 hours post incision 6.5 hours post 3.0 incision - 2/ H0587 Healing groin wound; Groin-Feb. 19, 1997 groin disease pCMVSport 7.5 hours post incision 3.0 H0590 Human adult small Human Adult Small Small Int. Uni-ZAP XR intestine, re-excision Intestine H0593 Olfactory Olfactory epithelium pCMVSport epithelium; nasalcavity from roof of left 3.0 nasal cacit H0597 Human Colon; re- Human Colon Lambda ZAP excision II H0599 Human Adult Heart; re- Human Adult Heart Heart Uni-ZAP XR excision H0604 Human Pituitary, re- Human Pituitary pBluescript excision H0606 Human Primary Breast Human Primary Breast disease Uni-ZAP XR Cancer; re-excision Breast Cancer H0615 Human Ovarian Cancer Ovarian Cancer Ovary disease Uni-ZAP XR Reexcision H0616 Human Testes, Human Testes Testis Uni-ZAP XR Reexcision H0617 Human Primary Breast Human Primary Breast disease Uni-ZAP XR Cancer Reexcision Breast Cancer H0618 Human Adult Testes, Human Adult Testis Testis Uni-ZAP XR Large Inserts, Reexcision H0619 Fetal Heart Human Fetal Heart Heart Uni-ZAP XR H0620 Human Fetal Kidney; Human Fetal Kidney Kidney Uni-ZAP XR Reexcision H0622 Human Pancreas Tumor; Human Pancreas Pancreas disease Uni-ZAP XR Reexcision Tumor H0624 12 Week Early Stage Twelve Week Old Embryo Uni-ZAP XR Human II; Reexcision Early Stage Human H0626 Saos2 Cells; Untreated Saos2 Cell Line; pSport1 Untreated H0632 Hepatocellular Hepatocellular Liver Lambda ZAP Tumor; re-excision Tumor II H0634 Human Testes Tumor, Human Testes Tumor Testis disease Uni-ZAP XR re-excision H0637 Dendritic Cells From Dentritic cells from pSport1 CD34 Cells CD34 cells H0638 CD40 activated CD40 activated pSport1 monocyte dendridic cells monocyte dendridic cells H0641 LPS activated derived LPS activated pSport1 dendritic cells monocyte derived dendritic cells H0643 Hep G2 Cells, PCR Hep G2 Cells Other library H0644 Human Placenta (re- Human Placenta Placenta Uni-ZAP XR excision) H0645 Fetal Heart, re-excision Human Fetal Heart Heart Uni-ZAP XR H0646 Lung, Cancer (4005313 Metastatic squamous pSport1 A3): Invasive Poorly cell lung carcinoma, Differentiated Lung poorly di Adenocarcinoma, H0647 Lung, Cancer (4005163 Invasive poorly disease pSport1 B7): Invasive, Poorly differentiated lung Diff. Adenocarcinoma, adenocarcinoma Metastatic H0648 Ovary, Cancer: Papillary Cstic disease pSport1 (4004562 B6) Papillary neoplasm of low Serous Cystic Neoplasm, malignant potentia Low Malignant Pot H0650 B-Cells B-Cells pCMVSport 3.0 H0651 Ovary, Normal: Normal Ovary pSport1 (9805C040R) H0652 Lung, Normal: (4005313 Normal Lung pSport1 B1) H0653 Stromal Cells Stromal Cells pSport1 H0654 Lung, Cancer: (4005313 Metastatic Squamous Other A3) Invasive Poorly- cell lung Carcinoma differentiated Metastatic poorly dif lung adenoc H0656 B-cells (unstimulated) B-cells pSport1 (unstimulated) H0657 B-cells (stimulated) B-cells (stimulated) pSport1 H0658 Ovary, Cancer 9809C332-Poorly Ovary & disease pSport1 (9809C332): Poorly differentiate Fallopian differentiated Tubes adenocarcinoma H0659 Ovary, Cancer Grade II Papillary Ovary disease pSport1 (15395A1F): Grade II Carcinoma, Ovary Papillary Carcinoma H0660 Ovary, Cancer: Poorly differentiated disease pSport1 (15799A1F) Poorly carcinoma, ovary differentiated carcinoma H0661 Breast, Cancer: Breast cancer disease pSport1 (4004943 A5) H0664 Breast, Cancer: Breast Cancer Breast disease pSport1 (9806C012R) H0670 Ovary, Cancer(4004650 Ovarian Cancer - pSport1 A3): Well- 4004650A3 Differentiated Micropapillary Serous Carcinoma H0672 Ovary, Cancer: Ovarian Ovary pSport1 (4004576 A8) Cancer(4004576A8) H0673 Human Prostate Cancer, Human Prostate Prostate Uni-ZAP XR Stage B2; re-excision Cancer, stage B2 H0675 Colon, Cancer: Colon Cancer pCMVSport (9808C064R) 9808C064R 3.0 H0676 Colon, Cancer: Colon Cancer pCMVSport (9808C064R)-total RNA 9808C064R 3.0 H0684 Serous Papillary Ovarian Cancer- Ovaries pCMVSport Adenocarcinoma 9810G606 3.0 H0686 Adenocarcinoma of Adenocarcinoma of pCMVSport Ovary, Human Cell Line Ovary, Human Cell 3.0 Line, # SW-626 H0687 Human normal Human normal Ovary pCMVSport ovary(#9610G215) ovary(#9610G215) 3.0 H0690 Ovarian Cancer, # Ovarian Cancer, pCMVSport 9702G001 #9702G001 3.0 H0695 mononucleocytes from mononucleocytes pCMVSport patient from patient at Shady 3.0 Grove Hospit N0004 Human Hippocampus Human Hippocampus S0003 Human Osteoclastoma Osteoclastoma bone disease Uni-ZAP XR S0010 Human Amygdala Amygdala Uni-ZAP XR S0011 STROMAL - Osteoclastoma bone disease Uni-ZAP XR OSTEOCLASTOMA S0013 Prostate Prostate prostate Uni-ZAP XR S0022 Human Osteoclastoma Osteoclastoma Uni-ZAP XR Stromal Cells - Stromal Cells unamplified S0026 Stromal cell TF274 stromal cell Bone Cell Line Uni-ZAP XR marrow S0027 Smooth muscle, serum Smooth muscle Pulmanary Cell Line Uni-ZAP XR treated artery S0028 Smooth muscle, control Smooth muscle Pulmanary Cell Line Uni-ZAP XR artery S0029 brain stem Brain stem brain Uni-ZAP XR S0031 Spinal cord Spinal cord spinal Uni-ZAP XR cord S0038 Human Whole Brain #2 - Human Whole Brain ZAP Express Oligo dT > 1.5 Kb #2 S0040 Adipocytes Human Adipocytes Uni-ZAP XR from Osteoclastoma S0044 Prostate BPH prostate BPH Prostate disease Uni-ZAP XR S0045 Endothelial cells-control Endothelial cell endothelial Cell Line Uni-ZAP XR cell-lung S0046 Endothelial-induced Endothelial cell endothelial Cell Line Uni-ZAP XR cell-lung S0049 Human Brain, Striatum Human Brain, Uni-ZAP XR Striatum S0051 Human Human disease Uni-ZAP XR Hypothalmus, Schizophrenia Hypothalamus, Schizophrenia S0052 neutrophils control human neutrophils blood Cell Line Uni-ZAP XR S0112 Hypothalamus Brain Uni-ZAP XR S0114 Anergic T-cell Anergic T-cell Cell Line Uni-ZAP XR S0116 Bone marrow Bone marrow Bone Uni-ZAP XR marrow S0126 Osteoblasts Osteoblasts Knee Cell Line Uni-ZAP XR S0142 Macrophage-oxLDL macrophage-oxidized blood Cell Line Uni-ZAP XR LDL treated S0144 Macrophage (GM-CSF Macrophage (GM- Uni-ZAP XR treated) CSF treated) S0152 PC3 Prostate cell line PC3 prostate cell line Uni-ZAP XR S0180 Bone Marrow Stroma, Bone Marrow disease Uni-ZAP XR TNF&LPS ind Stroma, TNF & LPS induced S0192 Synovial Fibroblasts Synovial Fibroblasts pSport1 (control) S0194 Synovial hypoxia Synovial Fibroblasts pSport1 S0196 Synovial IL-1/TNF Synovial Fibroblasts pSport1 stimulated S0206 Smooth Muscle- Smooth muscle Pulmanary Cell Line pBluescript HASTE normalized artery S0212 Bone Marrow Stromal Bone Marrow pSport1 Cell, untreated Stromal Cell, untreated S0218 Apoptotic T-cell, re- apoptotic cells Cell Line Uni-ZAP XR excision S0222 H. Frontal H. Brain, Frontal Brain disease Uni-ZAP XR cortex, epileptic; re- Cortex, Epileptic excision S0260 Spinal Cord, re-excision Spinal cord spinal Uni-ZAP XR cord S0276 Synovial hypoxia-RSF Synovial fobroblasts Synovial pSport1 subtracted (rheumatoid) tissue S0278 H Macrophage (GM- Macrophage (GM- Uni-ZAP XR CSF treated), re-excision CSF treated) S0280 Human Adipose Tissue, Human Adipose Uni-ZAP XR re-excision Tissue S0298 Bone marrow Bone marrow Bone pSport1 stroma, treated stroma, treatedSB marrow S0312 Human Human osteoarthritic disease pSport1 osteoarthritic; fraction II cartilage S0314 Human Human osteoarthritic disease pSport1 osteoarthritis; fraction I cartilage S0330 Palate normal Palate normal Uvula pSport1 S0344 Macrophage-oxLDL; re- macrophage-oxidized blood Cell Line Uni-ZAP XR excision LDL treated S0346 Human Amygdala; re- Amygdala Uni-ZAP XR excision S0354 Colon Normal II Colon Normal Colon pSport1 S0356 Colon Carcinoma Colon Carcinoma Colon disease pSport1 S0358 Colon Normal III Colon Normal Colon pSport1 S0360 Colon Tumor II Colon Tumor Colon disease pSport1 S0362 Human Gastrocnemius Gastrocnemius pSport1 muscle S0364 Human Quadriceps Quadriceps muscle pSport1 S0366 Human Soleus Soleus Muscle pSport1 S0374 Normal colon Normal colon pSport1 S0376 Colon Tumor Colon Tumor disease pSport1 S0378 Pancreas normal PCA4 Pancreas Normal pSport1 No PCA4 No S0380 Pancreas Tumor PCA4 Pancreas Tumor disease pSport1 Tu PCA4 Tu S0390 Smooth muscle, control; Smooth muscle Pulmanary Cell Line Uni-ZAP XR re-excision artery S0406 Rectum tumor Rectum tumor pSport1 S0408 Colon, normal Colon, normal pSport1 S0410 Colon, tumor Colon, tumor pSport1 S0418 CHME Cell Line; treated CHME Cell Line; pCMVSport 5 hrs treated 3.0 S0420 CHME Cell CHME Cell line, pSport1 Line, untreated untreatetd S0422 Mo7e Cell Line GM- Mo7e Cell Line GM- pCMVSport CSF treated (1 ng/ml) CSF treated (1 ng/ml) 3.0 S0426 Monocyte activated; re- Monocyte-activated blood Cell Line Uni-ZAP XR excision S0428 Neutrophils control; re- human neutrophils blood Cell Line Uni-ZAP XR excision S0434 Stomach Normal Stomach Normal disease pSport1 S0436 Stomach Tumor Stomach Tumor disease pSport1 S0438 Liver Normal Met5No Liver Normal pSport1 Met5No S0440 Liver Tumor Met 5 Tu Liver Tumor pSport1 S0442 Colon Normal Colon Normal pSport1 S0444 Colon Tumor Colon Tumor disease pSport1 S0450 Larynx Tumor Larynx Tumor pSport1 S0462 Thyroid Thyroiditis Thyroid Thyroiditis pSport1 S0474 Human blood platelets Platelets Blood Other platelets S3012 Smooth Muscle Serum Smooth muscle Pulmanary Cell Line pBluescript Treated, Norm artery S3014 Smooth muscle, serum Smooth muscle Pulmanary Cell Line pBluescript induced, re-exc artery S6024 Alzheimers, spongy Alzheimer's/Spongy Brain disease Uni-ZAP XR change change S6028 Human Manic Human Manic Brain disease Uni-ZAP XR Depression Tissue depression tissue T0006 Human Pineal Gland Human Pinneal pBluescript Gland SK- T0010 Human Infant Brain Human Infant Brain Other T0039 HSA 172 Cells Human HSA 172 cell pBluescript line SK- T0040 HSC172 Cells SA172 Cells pBluescript SK- T0041 Jurkat T-cell G1 phase Jurkat T-cell pBluescript SK- T0049 Aorta endothelial cells + Aorta endothelial pBluescript TNF-a cells SK- T0067 Human Thyroid Human Thyroid pBluescript SK- T0091 Liver, hepatocellular pBluescript carcinoma SK- T0109 Human (HCC) cell line pBluescript liver (mouse) metastasis, SK- remake L0163 Human heart cDNA heart (YNakamura) L0354 JG, Human foetal Bluescript Kidney tissue L0355 P, Human foetal Brain Bluescript Whole tissue L0361 Stratagene ovary ovary Bluescript SK (#937217) L0362 Stratagene ovarian Bluescript cancer (#937219) SK- L0367 NCI_CGAP_Sch1 Schwannoma tumor Bluescript SK- L0370 Johnston frontal cortex pooled frontal lobe brain Bluescript SK- L0371 NCI_CGAP_Br3 breast tumor breast Bluescript SK- L0372 NCI_CGAP_Co12 colon tumor colon Bluescript SK- L0373 NCI_CGAP_Co11 tumor colon Bluescript SK- L0374 NCI_CGAP_Co2 tumor colon Bluescript SK- L0375 NCI_CGAP_Kid6 kidney tumor kidney Bluescript SK- L0384 NCI_CGAP_Pr23 prostate tumor prostate Bluescript SK- L0438 normalized infant brain total brain brain lafmid BA cDNA L0439 Soares infant brain 1NIB whole Lafmid BA brain L0471 Human fetal heart, Lambda ZAP Lambda ZAP Express Express L0475 KG1-a Lambda Zap KG1-a Lambda Zap Express cDNA library Express (Stratagene) L0483 Human pancreatic islet Lambda ZAPII L0518 NCI_CGAP_Pr2 pAMP10 L0519 NCI_CGAP_Pr3 pAMP10 L0520 NCI_CGAP_Alv1 alveolar pAMP10 rhabdomyosarcoma L0521 NCI_CGAP_Ew1 Ewing's sarcoma pAMP10 L0529 NCI_CGAP_Pr6 prostate pAMP10 L0530 NCI_CGAP_Pr8 prostate pAMP10 L0581 Stratagene liver liver pBluescript (#937224) SK L0588 Stratagene endothelial pBluescript cell 937223 SK- L0591 Stratagene HeLa cell s3 pBluescript 937216 SK- L0592 Stratagene hNT neuron pBluescript (#937233) SK- L0593 Stratagene pBluescript neuroepithelium SK- (#937231) L0596 Stratagene colon colon pBluescript (#937204) SK- L0598 Morton Fetal Cochlea cochlea ear pBluescript SK- L0599 Stratagene lung lung pBluescript (#937210) SK- L0601 Stratagene pancreas pancreas pBluescript (#937208) SK- L0604 Stratagene muscle muscle skeletal pBluescript 937209 muscle SK- L0608 Stratagene lung lung carcinoma lung NCI-H69 pBluescript carcinoma 937218 SK- L0615 22 week old human fetal pBluescriptII liver cDNA library SK(−) L0622 HM1 pcDNAII (Invitrogen) L0623 HM3 pectoral muscle (after pcDNAII mastectomy) (Invitrogen) L0629 NCI_CGAP_Mel3 metastatic melanoma bowel pCMV- to bowel (skin SPORT4 primary) L0638 NCI_CGAP_Brn35 tumor, 5 pooled (see brain pCMV- description) SPORT6 L0640 NCI_CGAP_Br18 four pooled high- breast pCMV- grade tumors, SPORT6 including two prima L0641 NCI_CGAP_Co17 juvenile granulosa colon pCMV- tumor SPORT6 L0643 NCI_CGAP_Co19 moderately colon pCMV- differentiated SPORT6 adenocarcinoma L0644 NCI_CGAP_Co20 moderately colon pCMV- differentiated SPORT6 adenocarcinoma L0646 NCI_CGAP_Co14 moderately- colon pCMV- differentiated SPORT6 adenocarcinoma L0648 NCI_CGAP_Eso2 squamous cell esophagus pCMV- carcinoma SPORT6 L0649 NCI_CGAP_GU1 2 pooled high-grade genitourinary pCMV- transitional cell tract SPORT6 tumors L0652 NCI_CGAP_Lu27 four pooled poorly- lung pCMV- differentiated SPORT6 adenocarcinomas L0653 NCI_CGAP_Lu28 two pooled squamous lung pCMV- cell carcinomas SPORT6 L0655 NCI_CGAP_Lym12 lymphoma, follicular lymph pCMV- mixed small and node SPORT6 large cell L0657 NCI_CGAP_Ov23 tumor, 5 pooled (see ovary pCMV- description) SPORT6 L0659 NCI_CGAP_Pan1 adenocarcinoma pancreas pCMV- SPORT6 L0662 NCI_CGAP_Gas4 poorly differentiated stomach pCMV- adenocarcinoma with SPORT6 signet r L0663 NCI_CGAP_Ut2 moderately- uterus pCMV- differentiated SPORT6 endometrial adenocarcino L0664 NCI_CGAP_Ut3 poorly-differentiated uterus pCMV- endometrial SPORT6 adenocarcinoma, L0665 NCI_CGAP_Ut4 serous papillary uterus pCMV- carcinoma, high SPORT6 grade, 2 pooled t L0666 NCI_CGAP_Ut1 well-differentiated uterus pCMV- endometrial SPORT6 adenocarcinoma, 7 L0667 NCI_CGAP_CML1 myeloid cells, 18 whole pCMV- pooled CML cases, blood SPORT6 BCR/ABL rearra L0682 Stanley Frontal NB pool 2 frontal lobe (see brain pCR2.1- description) TOPO (Invitrogen) L0710 NIH_MGC_7 small cell carcinoma lung MGC3 pOTB7 L0717 Gessler Wilms tumor pSPORT1 L0731 Soares_pregnant_uterus_(—) uterus pT7T3-Pac NbHPU L0738 Human colorectal cancer pT7T3D L0740 Soares melanocyte melanocyte pT7T3D 2NbHM (Pharmacia) with a modified polylinker L0741 Soares adult brain brain pT7T3D N2b4HB55Y (Pharmacia) with a modified polylinker L0742 Soares adult brain brain pT7T3D N2b5HB55Y (Pharmacia) with a modified polylinker L0743 Soares breast 2NbHBst breast pT7T3D (Pharmacia) with a modified polylinker L0744 Soares breast 3NbHBst breast pT7T3D (Pharmacia) with a modified polylinker L0745 Soares retina N2b4HR retina eye pT7T3D (Pharmacia) with a modified polylinker L0746 Soares retina N2b5HR retina eye pT7T3D (Pharmacia) with a modified polylinker L0747 Soares_fetal_heart_NbHH19W heart pT7T3D (Pharmacia) with a modified polylinker L0748 Soares fetal liver spleen Liver and pT7T3D 1NFLS Spleen (Pharmacia) with a modified polylinker L0749 Soares_fetal_liver_spleen_(—) Liver and pT7T3D 1NFLS_S1 Spleen (Pharmacia) with a modified polylinker L0750 Soares_fetal_lung_NbHL19W lung pT7T3D (Pharmacia) with a modified polylinker L0751 Soares ovary tumor ovarian tumor ovary pT7T3D NbHOT (Pharmacia) with a modified polylinker L0752 Soares_parathyroid_tumor_(—) parathyroid tumor parathyroid pT7T3D NbHPA gland (Pharmacia) with a modified polylinker L0753 Soares_pineal_gland_N3HPG pineal pT7T3D gland (Pharmacia) with a modified polylinker L0754 Soares placenta Nb2HP placenta pT7T3D (Pharmacia) with a modified polylinker L0755 Soares_placenta_8to9weeks_(—) placenta pT7T3D 2NbHP8to9W (Pharmacia) with a modified polylinker L0756 Soares_multiple_sclerosis_(—) multiple sclerosis pT7T3D 2NbHMSP lesions (Pharmacia) with a modified polylinker V_TYPE L0757 Soares_senescent_fibroblasts_(—) senescent fibroblast pT7T3D NbHSF (Pharmacia) with a modified polylinker V_TYPE L0758 Soares_testis_NHT pT7T3D-Pac (Pharmacia) with a modified polylinker L0759 Soares_total_fetus_Nb2HF8_(—) pT7T3D-Pac 9w (Pharmacia) with a modified polylinker L0761 NCI_CGAP_CLL1 B-cell, chronic pT7T3D-Pac lymphotic leukemia (Pharmacia) with a modified polylinker L0763 NCI_CGAP_Br2 breast pT7T3D-Pac (Pharmacia) with a modified polylinker L0764 NCI_CGAP_Co3 colon pT7T3D-Pac (Pharmacia) with a modified polylinker L0766 NCI_CGAP_GCB1 germinal center B pT7T3D-Pac cell (Pharmacia) with a modified polylinker L0768 NCI_CGAP_GC4 pooled germ cell pT7T3D-Pac tumors (Pharmacia) with a modified polylinker L0769 NCI_CGAP_Brn25 anaplastic brain pT7T3D-Pac oligodendroglioma (Pharmacia) with a modified polylinker L0770 NCI_CGAP_Brn23 glioblastoma brain pT7T3D-Pac (pooled) (Pharmacia) with a modified polylinker L0771 NCI_CGAP_Co8 adenocarcinoma colon pT7T3D-Pac (Pharmacia) with a modified polylinker L0773 NCI_CGAP_Co9 colon tumor RER + colon pT7T3D-Pac (Pharmacia) with a modified polylinker L0774 NCI_CGAP_Kid3 kidney pT7T3D-Pac (Pharmacia) with a modified polylinker L0775 NCI_CGAP_Kid5 2 pooled tumors kidney pT7T3D-Pac (clear cell type) (Pharmacia) with a modified polylinker L0776 NCI_CGAP_Lu5 carcinoid lung pT7T3D-Pac (Pharmacia) with a modified polylinker L0777 Soares_NhHMPu_S1 Pooled human mixed pT7T3D-Pac melanocyte, fetal (see (Pharmacia) heart, and pregnant below) with a modified polylinker L0779 Soares_NFL_T_GBC_S1 pooled pT7T3D-Pac (Pharmacia) with a modified polylinker L0780 Soares_NSF_F8_9W_OT_(—) pooled pT7T3D-Pac PA_P_S1 (Pharmacia) with a modified polylinker L0782 NCI_CGAP_Pr21 normal prostate prostate pT7T3D-Pac (Pharmacia) with a modified polylinker L0783 NCI_CGAP_Pr22 normal prostate prostate pT7T3D-Pac (Pharmacia) with a modified polylinker L0784 NCI_CGAP_Lei2 leiomyosarcoma soft tissue pT7T3D-Pac (Pharmacia) with a modified polylinker L0785 Barstead spleen spleen pT7T3D-Pac HPLRB2 (Pharmacia) with a modified polylinker L0788 NCI_CGAP_Sub2 pT7T3D-Pac (Pharmacia) with a modified polylinker L0789 NCI_CGAP_Sub3 pT7T3D-Pac (Pharmacia) with a modified polylinker L0790 NCI_CGAP_Sub4 pT7T3D-Pac (Pharmacia) with a modified polylinker L0791 NCI_CGAP_Sub5 pT7T3D-Pac (Pharmacia) with a modified polylinker L0794 NCI_CGAP_GC6 pooled germ cell pT7T3D-Pac tumors (Pharmacia) with a modified polylinker L0800 NCI_CGAP_Co16 colon tumor, RER + colon pT7T3D-Pac (Pharmacia) with a modified polylinker L0803 NCI_CGAP_Kid11 kidney pT7T3D-Pac (Pharmacia) with a modified polylinker L0804 NCI_CGAP_Kid12 2 pooled tumors kidney pT7T3D-Pac (clear cell type) (Pharmacia) with a modified polylinker L0805 NCI_CGAP_Lu24 carcinoid lung pT7T3D-Pac (Pharmacia) with a modified polylinker L0809 NCI_CGAP_Pr28 prostate pT7T3D-Pac (Pharmacia) with a modified polylinker L2991 BN0264 breast_normal puc 18 L3811 NPC pituitary pBluescript sk(-) L3816 HEMBA1 whole embryo, pME18SFL3 mainly head L3825 NT2RM4 NT2 pME18SFL3 L3826 NT2RP1 NT2 pUC19FL3 L5565 NCI_CGAP_Brn66 glioblastoma with brain pCMV- probably TP53 SPORT6 mutation and witho L5566 NCI_CGAP_Brn70 anaplastic brain pCMV- oligodendroglioma SPORT6.ccdb L5622 NCI_CGAP_Skn3 skin pCMV- SPORT6 L5623 NCI_CGAP_Skn4 squamous cell skin pCMV- carcinoma SPORT6

[0468] TABLE 5 OMIM Reference Description 103581 Albright hereditary osteodystrophy-2 106300 Ankylosing spondylitis 108800 Atrial septal defect, secundum type 120290 OSMED syndrome, 215150 120290 Stickler syndrome, type II, 184840 120810 C4 deficiency 120820 C4 deficiency 142857 Pemphigoid, susceptibility to 142858 Beryllium disease, chronic, susceptibility to 146150 Hypomelanosis of Ito 150270 Laryngeal adductor paralysis 166800 Otosclerosis 167250 Paget disease of bone 170261 Bare lymphocyte syndrome, type I, due to TAP2 deficiency 176270 Prader-Willi syndrome 177900 Psoriasis susceptibility-1 179450 Ragweed sensitivity 192340 Diabetes insipidus, neurohypophyseal, 125700 201910 Adrenal hyperplasia, congenital, due to 21-hydroxylase deficiency 210900 Bloom syndrome 217000 C2 deficiency 218000 Andermann syndrome 222100 Diabetes mellitus, insulin-dependent-1 227220 [Eye color, brown] 233100 [Renal glucosuria] 234200 Neurodegeneration with brain iron accumulation 235200 Hemochromatosis 248611 Maple syrup urine disease, type Ib 256550 Sialidosis, type I 256550 Sialidosis, type II 600202 Dyslexia, specific, 2 600261 Ehlers-Danlos-like syndrome 601623 Angelman syndrome 601800 [Hair color, brown] 601868 Deafness, autosomal dominant 13 601889 Lymphoma, diffuse large cell 602117 Prader-Willi syndrome 602280 Retinitis pigmentosa-14, 600132 602475 Ossification of posterior longitudinal ligament of spine

[0469] The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

[0470] The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.

[0471] The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the secreted protein.

[0472] The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO: X, and/or a cDNA contained in ATCC deposit Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO: Y and/or a polypeptide encoded by the cDNA contained in ATCC deposit Z. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO: Y and/or a polypeptide sequence encoded by the cDNA contained in ATCC deposit Z are also encompassed by the invention.

[0473] Signal Sequences

[0474] The present invention also encompasses mature forms of the polypeptide having the polypeptide sequence of SEQ ID NO: Y and/or the polypeptide sequence encoded by the cDNA in a deposited clone. Polynucleotides encoding the mature forms (such as, for example, the polynucleotide sequence in SEQ ID NO: X and/or the polynucleotide sequence contained in the cDNA of a deposited clone) are also encompassed by the invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretary leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.

[0475] Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information from the residues surrounding the cleavage site, typically residues −13 to +2, where +1 indicates the amino terminus of the secreted protein. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. (von Heinje, supra.) However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.

[0476] In the present case, the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called SignalP (Henrik Nielsen et al., Protein Engineering 10:1-6 (1997)), which predicts the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated. The analysis of the amino acid sequences of the secreted proteins described herein by this program provided the results shown in Table 1A.

[0477] As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the present invention provides secreted polypeptides having a sequence shown in SEQ ID NO: Y which have an N-terminus beginning within 5 residues (i.e., +or −5 residues) of the predicted cleavage point. Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

[0478] Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. Nonetheless, the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ ID NO: X and/or the polynucleotide sequence contained in the cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as described below). These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

[0479] Polynucleotide and Polypeptide Variants

[0480] The present invention is directed to variants of the polynucleotide sequence disclosed in SEQ ID NO: X, the complementary strand thereto, and/or the cDNA sequence contained in a deposited clone.

[0481] The present invention also encompasses variants of the polypeptide sequence disclosed in SEQ ID NO: Y and/or encoded by a deposited clone.

[0482] “Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.

[0483] The present invention is also directed to nucleic acid molecules which comprise, or alternatively consist of, a nucleotide sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for example, the nucleotide coding sequence in SEQ ID NO: X or the complementary strand thereto, the nucleotide coding sequence contained in a deposited cDNA clone or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of SEQ ID NO: Y, a nucleotide sequence encoding the polypeptide encoded by the cDNA contained in a deposited clone, and/or polynucleotide fragments of any of these nucleic acid molecules (e.g., those fragments described herein).

[0484] Polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.

[0485] The present invention is also directed to polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example, the polypeptide sequence shown in SEQ ID NO: Y, the polypeptide sequence encoded by the cDNA contained in a deposited clone, and/or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein).

[0486] By a nucleic acid having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence shown in Table 1A, the ORF (open reading frame), or any fragment specified as described herein.

[0487] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the presence invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990)). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identiy are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the lenght of the subject nucleotide sequence, whichever is shorter.

[0488] If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.

[0489] For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.

[0490] By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

[0491] As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, an amino acid sequences shown in Table 1A (SEQ ID NO: Y) or to the amino acid sequence encoded by cDNA contained in a deposited clone can be determined conventionally using known computer programs. A preferred method for determing the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap-Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.

[0492] If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.

[0493] For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequnce are manually corrected for. No other manual corrections are to made for the purposes of the present invention.

[0494] The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).

[0495] Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

[0496] Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)

[0497] Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine EL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” (See, Abstract.) In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.

[0498] Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.

[0499] Thus, the invention further includes polypeptide variants which show substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.

[0500] The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.

[0501] The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.

[0502] As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

[0503] Besides conservative amino acid substitution, variants of the present invention include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification or (v) fusion of the polypeptide with another compound, such as albumin (including, but not limited to, recombinant albumin (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)). Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.

[0504] For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).)

[0505] A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of the present invention having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of the present invention, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In specific embodiments, the number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention or fragments thereof (e.g., the mature form and/or other fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions are preferable.

[0506] Polynucleotide and Polypeptide Fragments

[0507] The present invention is also directed to polynucleotide fragments of the polynucleotides of the invention.

[0508] In the present invention, a “polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence which: is a portion of that contained in a deposited clone, or encoding the polypeptide encoded by the cDNA in a deposited clone; is a portion of that shown in SEQ ID NO: X or the complementary strand thereto, or is a portion of a polynucleotide sequence encoding the polypeptide of SEQ ID NO: Y. The nucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in a deposited clone or the nucleotide sequence shown in SEQ ID NO: X. In this context “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred.

[0509] Moreover, representative examples, of polynucleotide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO: X, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.

[0510] In the present invention, a “polypeptide fragment” refers to an amino acid sequence which is a portion of that contained in SEQ ID NO: Y or encoded by the cDNA contained in a deposited clone. Protein (polypeptide) fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context “about” includes the particularly recited ranges or values, and ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.

[0511] Preferred polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form.

[0512] Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.

[0513] Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide fragments of SEQ ID NO: Y falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotides encoding these domains are also contemplated.

[0514] Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.

[0515] Preferably, the polynucleotide fragments of the invention encode a polypeptide which demonstrates a functional activity. By a polypeptide demonstrating a “functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) polypeptide of invention protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide of the invention for binding) to an antibody to the polypeptide of the invention], immunogenicity (ability to generate antibody which binds to a polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention.

[0516] The functional activity of polypeptides of the invention, and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.

[0517] For example, in one embodiment where one is assaying for the ability to bind or compete with full-length polypeptide of the invention for binding to an antibody of the polypeptide of the invention, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

[0518] In another embodiment, where a ligand for a polypeptide of the invention identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E., et al., 1995, Microbiol. Rev. 59:94-123. In another embodiment, physiological correlates of binding of a polypeptide of the invention to its substrates (signal transduction) can be assayed.

[0519] In addition, assays described herein (see Examples) and otherwise known in the art may routinely be applied to measure the ability of polypeptides of the invention and fragments, variants derivatives and analogs thereof to elicit related biological activity related to that of the polypeptide of the invention (either in vitro or in vivo). Other methods will be known to the skilled artisan and are within the scope of the invention.

[0520] Epitopes and Antibodies

[0521] The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO: Y, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO: X or contained in ATCC deposit No. Z under stringent hybridization conditions or lower stringency hybridization conditions as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO: X), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.

[0522] The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.

[0523] Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).

[0524] In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

[0525] Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).

[0526] Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.

[0527] As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention (e.g., those comprising an immunogenic or antigenic epitope) can be fused to heterologous polypeptide sequences. For example, polypeptides of the present invention (including fragments or variants thereof), may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof, resulting in chimeric polypeptides. By way of another non-limiting example, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused with albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)). In a preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with the mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0 322 094) which is herein incorporated by reference in its entirety. In another preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-x of human serum albumin, where x is an integer from 1 to 585 and the albumin fragment has human serum albumin activity. In another preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-z of human serum albumin, where z is an integer from 369 to 419, as described in U.S. Pat. No. 5,766,883 herein incorporated by reference in its entirety. Polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused to either the N- or C-terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide). Polynucleotides encoding fusion proteins of the invention are also encompassed by the invention.

[0528] Such fusion proteins as those described above may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.

[0529] Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308- 13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO: X and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

[0530] Antibodies

[0531] Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ ID NO: Y, and/or an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. In preferred embodiments, the immunoglobulin molecules of the invention are IgG1. In other preferred embodiments, the immunoglobulin molecules of the invention are IgG4.

[0532] Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (scFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.

[0533] The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).

[0534] Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.

[0535] Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10 ⁻¹² M, ¹⁰⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

[0536] The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.

[0537] Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptoriligand interactions with the polypeptides of the invention either partially or fully. Preferrably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.

[0538] The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).

[0539] Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).

[0540] As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

[0541] The antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

[0542] The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.

[0543] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0544] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples (e.g., Example 16). In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

[0545] Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.

[0546] Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

[0547] For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

[0548] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).

[0549] Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).

[0550] Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

[0551] Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

[0552] Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).

[0553] Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.

[0554] Polynucleotides Encoding Antibodies

[0555] The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO: Y.

[0556] The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0557] Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

[0558] Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties ), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

[0559] In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.

[0560] In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.

[0561] Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038-1041 (1988)).

[0562] Methods of Producing Antibodies

[0563] The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

[0564] Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.

[0565] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

[0566] A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).

[0567] In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0568] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

[0569] In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

[0570] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.

[0571] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

[0572] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.

[0573] The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

[0574] The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

[0575] Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.

[0576] The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.

[0577] The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341(1992) (said references incorporated by reference in their entireties).

[0578] As discussed, supra, the polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO: Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO: Y may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP A 232,262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).

[0579] Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

[0580] The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc.

[0581] Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

[0582] The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

[0583] Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

[0584] Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).

[0585] Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

[0586] An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.

[0587] Immunophenotyping

[0588] The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. The translation product of the gene of the present invention may be useful as a cell specific marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

[0589] These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.

[0590] Assays For Antibody Binding

[0591] The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).

[0592] Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0593] Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

[0594] ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

[0595] The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.

[0596] Therapeutic Uses

[0597] The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

[0598] A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

[0599] The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

[0600] The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.

[0601] It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10 ⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

[0602] Gene Therapy

[0603] In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.

[0604] Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.

[0605] For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0606] In a preferred aspect, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.

[0607] Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

[0608] In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO 93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).

[0609] In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

[0610] Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143- 155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used.

[0611] Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).

[0612] Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

[0613] In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.

[0614] The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

[0615] Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

[0616] In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

[0617] In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

[0618] In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.

[0619] Demonstration of Therapeutic or Prophylactic Activity

[0620] The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.

[0621] Therapeutic/Prophylactic Administration and Composition

[0622] The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.

[0623] Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.

[0624] Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0625] In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.

[0626] In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

[0627] In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

[0628] Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

[0629] In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

[0630] The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0631] In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0632] The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0633] The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0634] For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.

[0635] The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0636] Diagnosis and Imaging

[0637] Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.

[0638] The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

[0639] Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

[0640] One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

[0641] It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).

[0642] Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.

[0643] In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

[0644] Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.

[0645] In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).

[0646] Kits

[0647] The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).

[0648] In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.

[0649] In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.

[0650] In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.

[0651] In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, Mo.).

[0652] The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).

[0653] Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.

[0654] Fusion Proteins

[0655] Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because secreted proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.

[0656] Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.

[0657] Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.

[0658] Moreover, polypeptides of the present invention, including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).) Polynucleotides comprising or alternatively consisting of nucleic acids which encode these fusion proteins are also encompassed by the invention.

[0659] Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)

[0660] Moreover, the polypeptides of the present invention can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl, Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767 (1984).)

[0661] Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.

[0662] Vectors, Host Cells, and Protein Production

[0663] The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

[0664] The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

[0665] The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0666] As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

[0667] Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlbad, Calif.). Other suitable vectors will be readily apparent to the skilled artisan.

[0668] Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.

[0669] A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.

[0670] Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.

[0671] In one embodiment, the yeast Pichia pastoris is used to express the polypeptide of the present invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O₂. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O₂. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See, Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.

[0672] In one example, the plasmid vector pPIC9K is used to express DNA encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in “Pichia Protocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression vector allows expression and secretion of a protein of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.

[0673] Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.

[0674] In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.

[0675] In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination, resulting in the formation of a new transcription unit (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761, issued Mar. 31, 1998; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).

[0676] In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide sequence of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

[0677] The invention encompasses polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.

[0678] Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.

[0679] Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No.: 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

[0680] The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

[0681] As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.

[0682] The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

[0683] As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to a proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.

[0684] One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

[0685] As indicated above, pegylation of the proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.

[0686] One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.

[0687] Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.

[0688] The number of polyethylene glycol moieties attached to each protein of the invention (i.e., the degree of substitution) may also vary. For example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

[0689] The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

[0690] Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to the amino acid sequence of SEQ I) NO: Y or encoded by the cDNA contained in a deposited clone (including fragments, variants, splice variants, and fusion proteins, corresponding to these polypeptides as described herein). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.

[0691] As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.

[0692] Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence ( e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.

[0693] In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.

[0694] Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimenize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.

[0695] Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.

[0696] In another example, proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence. In a further embodiment, associations proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti-Flag® antibody.

[0697] The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

[0698] Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hyrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

[0699] Uses of the Polynucleotides

[0700] Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.

[0701] The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each polynucleotide of the present invention can be used as a chromosome marker.

[0702] Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NO: X. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ID NO: X will yield an amplified fragment.

[0703] Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, preselection by hybridization to construct chromosome specific-cDNA libraries and computer mapping techniques (See, e.g., Shuler, Trends Biotechnol 16:456-459 (1998) which is hereby incorporated by reference in its entirety).

[0704] Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. For a review of this technique, see Verna et al., “Human Chromosomes: a Manual of Basic Techniques,” Pergamon Press, New York (1988).

[0705] For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes).

[0706] The polynucleotides of the present invention would likewise be useful for radiation hybrid mapping, HAPPY mapping, and long range restriction mapping. For a review of these techniques and others known in the art, see, e.g., Dear, “Genome Mapping: A Practical Approach,” IRL Press at Oxford University Press, London (1997); Aydin, J. Mol. Med. 77:691-694 (1999); Hacia et al., Mol. Psychiatry 3:483-492 (1998); Herrick et al., Chromosome Res. 7:409-423 (1999); Hamilton et al., Methods Cell Biol. 62:265-280 (2000); and/or Ott, J. Hered. 90:68-70 (1999) each of which is hereby incorporated by reference in its entirety.

[0707] Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. (Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library).) Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.

[0708] Thus, once coinheritance is established, differences in the polynucleotide and the corresponding gene between affected and unaffected individuals can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal individuals is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide-can be used for further linkage analysis.

[0709] Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using polynucleotides of the present invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker.

[0710] Thus, the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an individual and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder.

[0711] In still another embodiment, the invention includes a kit for analyzing samples for the presence of proliferative and/or cancerous polynucleotides derived from a test subject. In a general embodiment, the kit includes at least one polynucleotide probe containing a nucleotide sequence that will specifically hybridize with a polynucleotide of the present invention and a suitable container. In a specific embodiment, the kit includes two polynucleotide probes defining an internal region of the polynucleotide of the present invention, where each probe has one strand containing a 31′mer-end internal to the region. In a further embodiment, the probes may be useful as primers for polymerase chain reaction amplification.

[0712] Where a diagnosis of a disorder, has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed polynucleotide of the present invention expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.

[0713] By “measuring the expression level of polynucleotide of the present invention” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the present invention or the level of the mRNA encoding the polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having a disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.

[0714] By “biological sample” is intended any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains the polypeptide of the present invention or mRNA. As indicated, biological samples include body fluids (such as semen, lymph, sera, plasma, urine, synovial fluid and spinal fluid) which contain the polypeptide of the present invention, and other tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.

[0715] The method(s) provided above may preferrably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides are attached to a solid support. In one exemplary method, the support may be a “gene chip” or a “biological chip” as described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip with polynucleotides of the present invention attached may be used to identify polymorphisms between the polynucleotide sequences, with polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e. their location, as well as, their existence) would be beneficial in identifying disease loci for many disorders, including cancerous diseases and conditions. Such a method is described in U.S. Pat. Nos. 5,858,659 and 5,856,104. The U.S. Patents referenced supra are hereby incorporated by reference in their entirety herein.

[0716] The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M. Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the strong binding. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.

[0717] The present invention is useful for detecting cancer in mammals. In particular the invention is useful during diagnosis of pathological cell proliferative neoplasias which include, but are not limited to: acute myelogenous leukemias including acute monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous leukemias including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.

[0718] Pathological cell proliferative diseases, disorders, and/or conditions are often associated with inappropriate activation of proto-oncogenes. (Gelmann, E. P. et al., “The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology,” in Neoplastic Diseases of the Blood, Vol 1., Wiernik, P. H. et al. eds., 161-182 (1985)). Neoplasias are now believed to result from the qualitative alteration of a normal cellular gene product, or from the quantitative modification of gene expression by insertion into the chromosome of a viral sequence, by chromosomal translocation of a gene to a more actively transcribed region, or by some other mechanism. (Gelmann et al., supra) It is likely that mutated or altered expression of specific genes is involved in the pathogenesis of some leukemias, among other tissues and cell types. (Gelmann et al., supra) Indeed, the human counterparts of the oncogenes involved in some animal neoplasias have been amplified or translocated in some cases of human leukemia and carcinoma. (Gelmann et al., supra)

[0719] For example, c-myc expression is highly amplified in the non-lymphocytic leukemia cell line HL-60. When HL-60 cells are chemically induced to stop proliferation, the level of c-myc is found to be downregulated. (International Publication Number WO 91/15580) However, it has been shown that exposure of HL-60 cells to a DNA construct that is complementary to the 5′ end of c-myc or c-myb blocks translation of the corresponding mRNAs which downregulates expression of the c-myc or c-myb proteins and causes arrest of cell proliferation and differentiation of the treated cells. (International Publication Number WO 91/15580; Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al., Proc. Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisan would appreciate the present invention's usefulness would not be limited to treatment of proliferative diseases, disorders, and/or conditions of hematopoietic cells and tissues, in light of the numerous cells and cell types of varying origins which are known to exhibit proliferative phenotypes.

[0720] In addition to the foregoing, a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). Both methods rely on binding of the polynucleotide to a complementary DNA or RNA. For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991) ) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat or prevent disease.

[0721] Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell.

[0722] The polynucleotides are also useful for identifying individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.

[0723] The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences. Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.

[0724] Forensic biology also benefits from using DNA-based identification techniques as disclosed herein. DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or surfactant, urine, fecal matter, etc., can be amplified using PCR. In one prior art technique, gene sequences amplified from polymorphic loci, such as DQa class II HLA gene, are used in forensic biology to identify individuals. (Erlich, H., PCR Technology, Freeman and Co. (1992).) Once these specific polymorphic loci are amplified, they are digested with one or more restriction enzymes, yielding an identifying set of bands on a Southern blot probed with DNA corresponding to the DQa class II I-LA gene. Similarly, polynucleotides of the present invention can be used as polymorphic markers for forensic purposes.

[0725] There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination.

[0726] In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to “subtract-out” known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a “gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.

[0727] Uses of the Polypeptides

[0728] Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.

[0729] A polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987).) Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.

[0730] In addition to assaying secreted protein levels in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.

[0731] A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131I, 112In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharnacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

[0732] Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

[0733] Moreover, polypeptides of the present invention can be used to treat, prevent, and/or diagnose disease. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).

[0734] Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat, prevent, and/or diagnose disease. For example, administration of an antibody directed to a polypeptide of the present invention can bind and reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).

[0735] At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the following biological activities.

[0736] Gene Therapy Methods

[0737] Another aspect of the present invention is to gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of a polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the invention that operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.

[0738] Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of the invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207-216 (1993); Ferrantini et al., Cancer Research, 53:107-1112 (1993); Ferrantini et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et al., Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy 4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38 (1996)), which are herein incorporated by reference. In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.

[0739] As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like). The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

[0740] In one embodiment, the polynucleotide of the invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.

[0741] The polynucleotide vector constructs of the invention used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEFI/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.

[0742] Any strong promoter known to those skilled in the art can be used for driving the expression of polynucleotide sequence of the invention. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter for the polynucleotides of the invention.

[0743] Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

[0744] The polynucleotide construct of the invention can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.

[0745] For the nakednucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.

[0746] The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.

[0747] The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called “gene guns”. These delivery methods are known in the art.

[0748] The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.

[0749] In certain embodiments, the polynucleotide constructs of the invention are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7416 (1987), which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA, 86:6077-6081 (1989), which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol. Chem., 265:10189-10192 (1990), which is herein incorporated by reference), in functional form.

[0750] Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl Acad. Sci. USA, 84:7413-7416 (1987), which is herein incorporated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

[0751] Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication NO: WO 90/11092 (which is herein incorporated by reference) for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g., Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.

[0752] Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

[0753] For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15 EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art.

[0754] The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology, 101:512-527 (1983), which-is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca²⁺-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilson et al., Cell, 17:77 (1979)); ether injection (Deamer et al., Biochim. Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad. Sci. USA, 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem., 255:10431 (1980); Szoka et al., Proc. Natl. Acad. Sci. USA, 75:145 (1978); Schaefer-Ridder et al., Science, 215:166 (1982)), which are herein incorporated by reference.

[0755] Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.

[0756] U.S. Pat. No.: 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incorporated by reference) provide methods for delivering DNA-cationic lipid complexes to mammals.

[0757] In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding polypeptides of the invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

[0758] The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy, 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO₄ precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

[0759] The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding polypeptides of the invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express polypeptides of the invention.

[0760] In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotides of the invention contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses polypeptides of the invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartzet al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1-antitrypsin and CFRR to the lungs of cotton rats (Rosenfeld et al.,Science, 252:431-434 (1991); Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA, 76:6606 (1979)).

[0761] Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel., 3:499-503 (1993); Rosenfeld et al., Cell, 68:143-155 (1992); Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al., Nature Genet., 7:362-369 (1994); Wilson et al., Nature, 365:691-692 (1993); and U.S. Pat. No.: 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the E1 region of adenovirus and constitutively express E1a and E1b, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

[0762] Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

[0763] In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol., 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

[0764] For example, an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct containing polynucleotides of the invention is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct of the invention. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express the desired gene product.

[0765] Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding the polypeptide sequence of interest) via homologous recombination (see, e.g., U.S. Pat. No.: 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.

[0766] Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5′ end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.

[0767] The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter. The amplified promoter and targeting sequences are digested and ligated together.

[0768] The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.

[0769] The promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence,

[0770] The polynucleotides encoding polypeptides of the present invention may be administered along with other polynucleotides encoding other angiongenic proteins. Angiogenic proteins include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.

[0771] Preferably, the polynucleotide encoding a polypeptide of the invention contains a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5′ end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.

[0772] Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (i.e., “gene guns”), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers. (Kaneda et al., Science, 243:375 (1989)).

[0773] A preferred method of local administration is by direct injection. Preferably, a recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries. Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.

[0774] Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.

[0775] Therapeutic compositions useful in systemic administration, include recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site.

[0776] Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA, 189:11277-11281(1992), which is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.

[0777] Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian. Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly

[0778] Biological Activities

[0779] The polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists could be used to treat the associated disease.

[0780] Polynucleotides, translation products and antibodies corresponding to this gene may be useful for the diagnosis, prognosis, prevention, and/or treatment of diseases and/or disorders associated with the following systems.

[0781] Immune Activity

[0782] Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing and/or prognosing diseases, disorders, and/or conditions of the immune system, by, for example, activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.

[0783] In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to treat diseases and disorders of the immune system and/or to inhibit or enhance an immune response generated by cells associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1A, column 8 (Tissue Distribution Library Code).

[0784] Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing, and/or prognosing immunodeficiencies, including both congenital and acquired immunodeficiencies. Examples of B cell immunodeficiencies in which immunoglobulin levels B cell function and/or B cell numbers are decreased include: X-linked agammaglobulinemia (Bruton's disease), X-linked infantile agammaglobulinemia, X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, X-linked lymphoproliferative syndrome (XLP), agammaglobulinemia including congenital and acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, unspecified hypogammaglobulinemia, recessive agammaglobulinemia (Swiss type), Selective IgM deficiency, selective IgA deficiency, selective IgG subclass deficiencies, IgG subclass deficiency (with or without IgA deficiency), Ig deficiency with increased IgM, IgG and IgA deficiency with increased IgM, antibody deficiency with normal or elevated Igs, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), common variable immunodeficiency (CVID), common variable immunodeficiency (CVI) (acquired), and transient hypogammaglobulinemia of infancy.

[0785] In specific embodiments, ataxia-telangiectasia or conditions associated with ataxia-telangiectasia are treated, prevented, diagnosed, and/or prognosing using the polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof.

[0786] Examples of congenital immunodeficiencies in which T cell and/or B cell function and/or number is decreased include, but are not limited to: DiGeorge anomaly, severe combined immunodeficiencies (SCID) (including, but not limited to, X-linked SCID, autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare lymphocyte syndrome), Wiskott-Aldrich syndrome, and ataxia telangiectasia), thymic hypoplasia, third and fourth pharyngeal pouch syndrome, 22q11.2 deletion, chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4+T-lymphocytopenia, immunodeficiency with predominant T cell defect (unspecified), and unspecified immunodeficiency of cell mediated immunity.

[0787] In specific embodiments, DiGeorge anomaly or conditions associated with DiGeorge anomaly are treated, prevented, diagnosed, and/or prognosed using polypeptides or polynucleotides of the invention, or antagonists or agonists thereof.

[0788] Other immunodeficiencies that may be treated, prevented, diagnosed, and/or prognosed using polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof, include, but are not limited to, chronic granulomatous disease, Chédiak-Figashi syndrome, myeloperoxidase deficiency, leukocyte glucose-6-phosphate dehydrogenase deficiency, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency, complement component deficiencies (including C1, C2, C3, C4, C5, C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic alymphoplasia-aplasia, immunodeficiency with thymoma, severe congenital leukopenia, dysplasia with immunodeficiency, neonatal neutropenia, short limbed dwarfism, and Nezelof syndrome-combined immunodeficiency with Igs.

[0789] In a preferred embodiment, the immunodeficiencies and/or conditions associated with the immunodeficiencies recited above are treated, prevented, diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.

[0790] In a preferred embodiment polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among immunodeficient individuals. In specific embodiments, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among B cell and/or T cell immunodeficient individuals.

[0791] The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing and/or prognosing autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of polynucleotides and polypeptides of the invention that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune disorders.

[0792] Autoimmune diseases or disorders that may be treated, prevented, diagnosed and/or prognosed by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, one or more of the following: systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia purpura, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, purpura (e.g., Henloch-Scoenlein purpura), autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), and insulin-resistant diabetes mellitus.

[0793] Additional disorders that are likely to have an autoimmune component that may be treated, prevented, and/or diagnosed with the compositions of the invention include, but are not limited to, type II collagen-induced arthritis, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff-Man Syndrome, autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye disorders.

[0794] Additional disorders that are likely to have an autoimmune component that may be treated, prevented, diagnosed and/or prognosed with the compositions of the invention include, but are not limited to, scleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by antispermatozoal antibodies), glomerulonephritis (often characterized, e.g., by glomerular basement membrane antibodies or immune complexes), bullous pemphigoid (often characterized, e.g., by IgG and complement in basement membrane), Sjogren's syndrome (often characterized, e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes mellitus (often characterized, e.g., by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies).

[0795] Additional disorders that may have an autoimmune component that may be treated, prevented, diagnosed and/or prognosed with the compositions of the invention include, but are not limited to, chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitochondria antibodies), other endocrine gland failure (often characterized, e.g., by specific tissue antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to IgE), asthma (often characterized, e.g., by IgG and IgM antibodies to IgE), and many other inflammatory, granulomatous, degenerative, and atrophic disorders.

[0796] In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using for example, antagonists or agonists, polypeptides or polynucleotides, or antibodies of the present invention. In a specific preferred embodiment, rheumatoid arthritis is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.

[0797] In another specific preferred embodiment, systemic lupus erythematosus is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention. In another specific preferred embodiment, idiopathic thrombocytopenia purpura is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.

[0798] In another specific preferred embodiment IgA nephropathy is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.

[0799] In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention

[0800] In preferred embodiments, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a immunosuppressive agent(s).

[0801] Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, prognosing, and/or diagnosing diseases, disorders, and/or conditions of hematopoietic cells. Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells, including but not limited to, leukopenia, neutropenia, anemia, and thrombocytopenia. Alternatively, Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with an increase in certain (or many) types of hematopoietic cells, including but not limited to, histiocytosis.

[0802] Allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, prevented, diagnosed and/or prognosed using polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof. Moreover, these molecules can be used to treat, prevent, prognose, and/or diagnose anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.

[0803] Additionally, polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof, may be used to treat, prevent, diagnose and/or prognose IgE-mediated allergic reactions. Such allergic reactions include, but are not limited to, asthma, rhinitis, and eczema. In specific embodiments, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to modulate IgE concentrations in vitro or in vivo.

[0804] Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention have uses in the diagnosis, prognosis, prevention, and/or treatment of inflammatory conditions. For example, since polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists of the invention may inhibit the activation, proliferation and/or differentiation of cells involved in an inflammatory response, these molecules can be used to prevent and/or treat chronic and acute inflammatory conditions. Such inflammatory conditions include, but are not limited to, for example, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome), ischemia-reperfusion injury, endotoxin lethality, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, over production of cytokines (e.g., TNF or IL-1.), respiratory disorders (e.g., asthma and allergy); gastrointestinal disorders (e.g., inflammatory bowel disease); cancers (e.g., gastric, ovarian, lung, bladder, liver, and breast); CNS disorders (e.g., multiple sclerosis; ischemic brain injury and/or stroke, traumatic brain injury, neurodegenerative disorders (e.g., Parkinson's disease and Alzheimer's disease); AIDS-related dementia; and prion disease); cardiovascular disorders (e.g., atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary bypass complications); as well as many additional diseases, conditions, and disorders that are characterized by inflammation (e.g., hepatitis, rheumatoid arthritis, gout, trauma, pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusion injury, Grave's disease, systemic lupus erythematosus, diabetes mellitus, and allogenic transplant rejection).

[0805] Because inflammation is a fundamental defense mechanism, inflammatory disorders can effect virtually any tissue of the body. Accordingly, polynucleotides, polypeptides, and antibodies of the invention, as well as agonists or antagonists thereof, have uses in the treatment of tissue-specific inflammatory disorders, including, but not limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, media otitis, meningitis, metritis, mucitis, myocarditis, myosititis, myringitis, nephritis, neuritis, orchitis, osteochondritis, otitis, pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis, salpingitis, scleritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis, steatitis, stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis, and vaginitis.

[0806] In specific embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to diagnose, prognose, prevent, and/or treat organ transplant rejections and graft-versus-host disease. Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. Polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD. In specific embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing experimental allergic and hyperacute xenograft rejection.

[0807] In other embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to diagnose, prognose, prevent, and/or treat immune complex diseases, including, but not limited to, serum sickness, post streptococcal glomerulonephritis, polyarteritis nodosa, and immune complex-induced vasculitis.

[0808] Polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the invention can be used to treat, detect, and/or prevent infectious agents. For example, by increasing the immune response, particularly increasing the proliferation activation and/or differentiation of B and/or T cells, infectious diseases may be treated, detected, and/or prevented. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may also directly inhibit the infectious agent (refer to section of application listing infectious agents, etc), without necessarily eliciting an immune response.

[0809] In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a vaccine adjuvant that enhances immune responsiveness to an antigen. In a specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance tumor-specific immune responses.

[0810] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-viral immune responses. Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant, include virus and virus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIV/AIDS, respiratory syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpes simplex, and yellow fever.

[0811] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant, include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B.

[0812] In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, and Borrelia burgdoferi.

[0813] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-parasitic immune responses. Anti-parasitic immune responses that may be enhanced using the compositions of the invention as an adjuvant, include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria) or Leishmania.

[0814] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed to treat infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis; for example, by preventing the recruitment and activation of mononuclear phagocytes.

[0815] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an antigen for the generation of antibodies to inhibit or enhance immune mediated responses against polypeptides of the invention.

[0816] In one embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are administered to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human) to boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production and immunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.

[0817] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a stimulator of B cell responsiveness to pathogens.

[0818] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an activator of T cells.

[0819] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent that elevates the immune status of an individual prior to their receipt of immunosuppressive therapies.

[0820] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to induce higher affinity antibodies.

[0821] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to increase serum immunoglobulin concentrations.

[0822] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to accelerate recovery of immunocompromised individuals.

[0823] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among aged populations and/or neonates.

[0824] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an immune system enhancer prior to, during, or after bone marrow transplant and/or other transplants (e.g., allogeneic or xenogeneic organ transplantation). With respect to transplantation, compositions of the invention may be administered prior to, concomitant with, and/or after transplantation. In a specific embodiment, compositions of the invention are administered after transplantation, prior to the beginning of recovery of T-cell populations. In another specific embodiment, compositions of the invention are first administered after transplantation after the beginning of recovery of T cell populations, but prior to full recovery of B cell populations.

[0825] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among individuals having an acquired loss of B cell function. Conditions resulting in an acquired loss of B cell function that may be ameliorated or treated by administering the polypeptides, antibodies, polynucleotides and/or agonists or antagonists thereof, include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).

[0826] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency. Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the polypeptides, antibodies, polynucleotides and/or agonists or antagonists thereof, include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, and recovery from surgery.

[0827] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a regulator of antigen presentation by monocytes, dendritic cells, and/or B-cells. In one embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention enhance antigen presentation or antagonizes antigen presentation in vitro or in vivo. Moreover, in related embodiments, said enhancement or antagonism of antigen presentation may be useful as an anti-tumor treatment or to modulate the immune system.

[0828] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to direct an individual's immune system towards development of a humoral response (i.e. TH2) as opposed to a TH1 cellular response.

[0829] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means to induce tumor proliferation and thus make it more susceptible to anti-neoplastic agents. For example, multiple myeloma is a slowly dividing disease and is thus refractory to virtually all anti-neoplastic regimens. If these cells were forced to proliferate more rapidly their susceptibility profile would likely change.

[0830] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a stimulator of B cell production in pathologies such as AIDS, chronic lymphocyte disorder and/or Common Variable Immunodificiency.

[0831] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for generation and/or regeneration of lymphoid tissues following surgery, trauma or genetic defect. In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used in the pretreatment of bone marrow samples prior to transplant.

[0832] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a gene-based therapy for genetically inherited disorders resulting in immuno-incompetence/immunodeficiency such as observed among SCID patients.

[0833] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of activating monocytes/macrophages to defend against parasitic diseases that effect monocytes such as Leishmania.

[0834] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of regulating secreted cytokines that are elicited by polypeptides of the invention.

[0835] In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used in one or more of the applications decribed herein, as they may apply to veterinary medicine.

[0836] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of blocking various aspects of immune responses to foreign agents or self. Examples of diseases or conditions in which blocking of certain aspects of immune responses may be desired include autoimmune disorders such as lupus, and arthritis, as well as immunoresponsiveness to skin allergies, inflammation, bowel disease, injury and diseases/disorders associated with pathogens.

[0837] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for preventing the B cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and multiple sclerosis.

[0838] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a inhibitor of B and/or T cell migration in endothelial cells. This activity disrupts tissue architecture or cognate responses and is useful, for example in disrupting immune responses, and blocking sepsis.

[0839] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for chronic hypergammaglobulinemia evident in such diseases as monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's disease, related idiopathic monoclonal gammopathies, and plasmacytomas.

[0840] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed for instance to inhibit polypeptide chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and natural killer cells, in certain autoimmune and chronic inflammatory and infective diseases. Examples of autoimmune diseases are described herein and include multiple sclerosis, and insulin-dependent diabetes.

[0841] The polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed to treat idiopathic hyper-eosinophilic syndrome by, for example, preventing eosinophil production and migration.

[0842] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used to enhance or inhibit complement mediated cell lysis.

[0843] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used to enhance or inhibit antibody dependent cellular cytotoxicity.

[0844] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed for treating atherosclerosis, for example, by preventing monocyte infiltration in the artery wall.

[0845] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed to treat adult respiratory distress syndrome (ARDS).

[0846] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be useful for stimulating wound and tissue repair, stimulating angiogenesis, and/or stimulating the repair of vascular or lymphatic diseases or disorders. Additionally, agonists and antagonists of the invention may be used to stimulate the regeneration of mucosal surfaces.

[0847] In a specific embodiment, polynucleotides or polypeptides, and/or agonists thereof are used to diagnose, prognose, treat, and/or prevent a disorder characterized by primary or acquired immunodeficiency, deficient serum immunoglobulin production, recurrent infections, and/or immune system dysfunction. Moreover, polynucleotides or polypeptides, and/or agonists thereof may be used to treat or prevent infections of the joints, bones, skin, and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but not limited to, CVID, other primary immune deficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), and/or pneumocystis carnii. Other diseases and disorders that may be prevented, diagnosed, prognosed, and/or treated with polynucleotides or polypeptides, and/or agonists of the present invention include, but are not limited to, HIV infection, HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunction anemia, thrombocytopenia, and hemoglobinuria.

[0848] In another embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention are used to treat, and/or diagnose an individual having common variable immunodeficiency disease (“CVID”; also known as “acquired agammaglobulinemia” and “acquired hypogammaglobulinemia”) or a subset of this disease.

[0849] In a specific embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to diagnose, prognose, prevent, and/or treat cancers or neoplasms including immune cell or immune tissue-related cancers or neoplasms. Examples of cancers or neoplasms that may be prevented, diagnosed, or treated by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL) Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, EBV-transformed diseases, and/or diseases and disorders described in the section entitled “Hyperproliferative Disorders” elsewhere herein.

[0850] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for decreasing cellular proliferation of Large B-cell Lymphomas.

[0851] In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of decreasing the involvement of B cells and Ig associated with Chronic Myelogenous Leukemia.

[0852] In specific embodiments, the compositions of the invention are used as an agent to boost immunoresponsiveness among B cell immunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy.

[0853] Antagonists of the invention include, for example, binding and/or inhibitory antibodies, antisense nucleic acids, ribozymes or soluble forms of the polypeptides of the present invention (e.g., Fc fusion protein; see, e.g., Example 9). Agonists of the invention include, for example, binding or stimulatory antibodies, and soluble forms of the polypeptides (e.g., Fc fusion proteins; see, e.g., Example 9). polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present-invention may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as described herein.

[0854] In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are administered to an animal (including, but not limited to, those listed above, and also including transgenic animals) incapable of producing functional endogenous antibody molecules or having an otherwise compromised endogenous immune system, but which is capable of producing human immunoglobulin molecules by means of a reconstituted or partially reconstituted immune system from another animal (see, e.g., published PCT Application Nos. WO98/24893, WO/9634096, WO/9633735, and WO/9110741). Administration of polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention to such animals is useful for the generation of monoclonal antibodies against the polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention in an organ system listed above.

[0855] Blood-Related Disorders

[0856] The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to modulate hemostatic (the stopping of bleeding) or thrombolytic (clot dissolving) activity. For example, by increasing hemostatic or thrombolytic activity, polynucleotides or polypeptides, and/or agonists or antagonists of the present invention could be used to treat or prevent blood coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia, factor deficiencies, hemophilia), blood platelet diseases, disorders, and/or conditions (e.g., thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment or prevention of heart attacks (infarction), strokes, or scarring.

[0857] In specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to prevent, diagnose, prognose, and/or treat thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. In specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used for the prevention of occulsion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atrial fibrillation, for reducing the risk of embolism associated with mechanical heart valves and or mitral valves disease. Other uses for the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention, include, but are not limited to, the prevention of occlusions in extrcorporeal devices (e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).

[0858] In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to prevent, diagnose, prognose, and/or treat diseases and disorders of the blood and/or blood forming organs associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1A, column 8 (Tissue Distribution Library Code).

[0859] The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to modulate hematopoietic activity (the formation of blood cells). For example, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to increase the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the prevention, detection, diagnosis and/or treatment of anemias and leukopenias described below. Alternatively, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to decrease the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the prevention, detection, diagnosis and/or treatment of leukocytoses, such as, for example eosinophilia.

[0860] The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to prevent, treat, or diagnose blood dyscrasia.

[0861] Anemias are conditions in which the number of red blood cells or amount of hemoglobin (the protein that carries oxygen) in them is below normal. Anemia may be caused by excessive bleeding, decreased red blood cell production, or increased red blood cell destruction (hemolysis). The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing anemias. Anemias that may be treated prevented or diagnosed by the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include iron deficiency anemia, hypochromic anemia, microcytic anemia, chlorosis, hereditary siderob;astic anemia, idiopathic acquired sideroblastic anemia, red cell aplasia, megaloblastic anemia (e.g., pernicious anemia, (vitamin B12 deficiency) and folic acid deficiency anemia), aplastic anemia, hemolytic anemias (e.g., autoimmune helolytic anemia, microangiopathic hemolytic anemia, and paroxysmal nocturnal hemoglobinuria). The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing anemias associated with diseases including but not limited to, anemias associated with systemic lupus erythematosus, cancers, lymphomas, chronic renal disease, and enlarged spleens. The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing anemias arising from drug treatments such as anemias associated with methyldopa, dapsone, and/or sulfadrugs. Additionally, rhe polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing anemias associated with abnormal red blood cell architecture including but not limited to, hereditary spherocytosis, hereditary elliptocytosis, glucose-6-phosphate dehydrogenase deficiency, and sickle cell anemia.

[0862] The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing hemoglobin abnormalities, (e.g., those associated with sickle cell anemia, hemoglobin C disease, hemoglobin S—C disease, and hemoglobin E disease). Additionally, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating thalassemias, including, but not limited to major and minor forms of alpha-thalassemia and beta-thalassemia.

[0863] In another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating bleeding disorders including, but not limited to, thrombocytopenia (e.g., idiopathic thrombocytopenic purpura, and thrombotic thrombocytopenic purpura), Von Willebrand's disease, hereditary platelet disorders (e.g., storage pool disease such as Chediak-Higashi and Hermansky-Pudlak syndromes, thromboxane A2 dysfunction, thromboasthenia, and Bernard-Soulier syndrome), hemolytic-uremic syndrome, hemophelias such as hemophelia A or Factor VII deficiency and Christmas disease or Factor IX deficiency, Hereditary Hemorhhagic Telangiectsia, also known as Rendu-Osler-Weber syndrome, allergic purpura (Henoch Schonlein purpura) and disseminated intravascular coagulation.

[0864] The effect of the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention on the clotting time of blood may be monitored using any of the clotting tests known in the art including, but not limited to, whole blood partial thromboplastin time (PTT), the activated partial thromboplastin time (aPTT), the activated clotting time (ACT), the recalcified activated clotting time, or the Lee-White Clotting time.

[0865] Several diseases and a variety of drugs can cause platelet dysfunction. Thus, in a specific embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating acquired platelet dysfunction such as platelet dysfunction accompanying kidney failure, leukemia, multiple myeloma, cirrhosis of the liver, and systemic lupus erythematosus as well as platelet dysfunction associated with drug treatments, including treatment with aspirin, ticlopidine, nonsteroidal anti-inflammatory drugs (used for arthritis, pain, and sprains), and penicillin in high doses.

[0866] In another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders characterized by or associated with increased or decreased numbers of white blood cells. Leukopenia occurs when the number of white blood cells decreases below normal. Leukopenias include, but are not limited to, neutropenia and lymphocytopenia. An increase in the number of white blood cells compared to normal is known as leukocytosis. The body generates increased numbers of white blood cells during infection. Thus, leukocytosis may simply be a normal physiological parameter that reflects infection. Alternatively, leukocytosis may be an indicator of injury or other disease such as cancer. Leokocytoses, include but are not limited to, eosinophilia, and accumulations of macrophages. In specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating leukopenia. In other specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating leukocytosis.

[0867] Leukopenia may be a generalized decreased in all types of white blood cells, or may be a specific depletion of particular types of white blood cells. Thus, in specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating decreases in neutrophil numbers, known as neutropenia. Neutropenias that may be diagnosed, prognosed, prevented, and/or treated by the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, infantile genetic agranulocytosis, familial neutropenia, cyclic neutropenia, neutropenias resulting from or associated with dietary deficiencies (e.g., vitamin B 12 deficiency or folic acid deficiency), neutropenias resulting from or associated with drug treatments (e.g., antibiotic regimens such as penicillin treatment, sulfonamide treatment, anticoagulant treatment, anticonvulsant drugs, anti-thyroid drugs, and cancer chemotherapy), and neutropenias resulting from increased neutrophil destruction that may occur in association with some bacterial or viral infections, allergic disorders, autoimmune diseases, conditions in which an individual has an enlarged spleen (e.g., Felty syndrome, malaria and sarcoidosis), and some drug treatment regimens.

[0868] The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating lymphocytopenias (decreased numbers of B and/or T lymphocytes), including, but not limited lymphocytopenias resulting from or associated with stress, drug treatments (e.g., drug treatment with corticosteroids, cancer chemotherapies, and/or radiation therapies), AIDS infection and/or other diseases such as, for example, cancer, rheumatoid arthritis, systemic lupus erythematosus, chronic infections, some viral infections and/or hereditary disorders (e.g., DiGeorge syndrome, Wiskott-Aldrich Syndome, severe combined immunodeficiency, ataxia telangiectsia).

[0869] The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders associated with macrophage numbers and/or macrophage function including, but not limited to, Gaucher's disease, Niemann-Pick disease, Letterer-Siwe disease and Hand-Schuller-Christian disease.

[0870] In another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders associated with eosinophil numbers and/or eosinophil function including, but not limited to, idiopathic hypereosinophilic syndrome, eosinophilia-myalgia syndrome, and Hand-Schuller-Christian disease.

[0871] In yet another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating leukemias and lymphomas including, but not limited to, acute lymphocytic (lymphpblastic) leukemia (ALL), acute myeloid (myelocytic, myelogenous, myeloblastic, or myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., B cell leukemias, T cell leukemias, Sezary syndrome, and Hairy cell leukenia), chronic myelocytic (myeloid, myelogenous, or granulocytic) leukemia, Hodgkin's lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, and mycosis fungoides.

[0872] In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders of plasma cells including, but not limited to, plasma cell dyscrasias, monoclonal gammaopathies, monoclonal gammopathies of undetermined significance, multiple myeloma, macroglobulinemia, Waldenstrom's macroglobulinemia, cryoglobulinemia, and Raynaud's phenomenon.

[0873] In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing myeloproliferative disorders, including but not limited to, polycythemia vera, relative polycythemia, secondary polycythemia, myelofibrosis, acute myelofibrosis, agnogenic myelod metaplasia, thrombocythemia, (including both primary and seconday thrombocythemia) and chronic myelocytic leukemia.

[0874] In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as a treatment prior to surgery, to increase blood cell production.

[0875] In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to enhance the migration, phagocytosis, superoxide production, antibody dependent cellular cytotoxicity of neutrophils, eosionophils and macrophages.

[0876] In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to increase the number of stem cells in circulation prior to stem cells pheresis. In another specific embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to increase the number of stem cells in circulation prior to platelet pheresis.

[0877] In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to increase cytokine production.

[0878] In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in preventing, diagnosing, and/or treating primary hematopoietic disorders.

[0879] Hyperproliferative Disorders

[0880] In certain embodiments, polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used to treat or detect hyperproliferative disorders, including neoplasms. Polynucleotides or polypeptides, or agonists or antagonists of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, Polynucleotides or polypeptides, or agonists or antagonists of the present invention may proliferate other cells which can inhibit the hyperproliferative disorder.

[0881] For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative disorders can be treated. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent.

[0882] Examples of hyperproliferative disorders that can be treated or detected by polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to neoplasms located in the: colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.

[0883] Similarly, other hyperproliferative disorders can also be treated or detected by polynucleotides or polypeptides, or agonists or antagonists of the present invention. Examples of such hyperproliferative disorders include, but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic, Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.

[0884] In another preferred embodiment, polynucleotides or polypeptides, or agonists or antagonists of the present invention are used to diagnose, prognose, prevent, and/or treat premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above. Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79.)

[0885] Hyperplasia is a form of controlled cell proliferation, involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. Hyperplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, angiofollicular mediastinal lymph node hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical melanocytic hyperplasia, basal cell hyperplasia, benign giant lymph node hyperplasia, cementum hyperplasia, congenital adrenal hyperplasia, congenital sebaceous hyperplasia, cystic hyperplasia, cystic hyperplasia of the breast, denture hyperplasia, ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia, focal epithelial hyperplasia, gingival hyperplasia, inflammatory fibrous hyperplasia, inflammatory papillary hyperplasia, intravascular papillary endothelial hyperplasia, nodular hyperplasia of prostate, nodular regenerative hyperplasia, pseudoepitheliomatous hyperplasia, senile sebaceous hyperplasia, and verrucous hyperplasia.

[0886] Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, agnogenic myeloid metaplasia, apocrine metaplasia, atypical metaplasia, autoparenchymatous metaplasia, connective tissue metaplasia, epithelial metaplasia, intestinal metaplasia, metaplastic anemia, metaplastic ossification, metaplastic polyps, myeloid metaplasia, primary myeloid metaplasia, secondary myeloid metaplasia, squamous metaplasia, squamous metaplasia of amnion, and symptomatic myeloid metaplasia.

[0887] Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation. Dysplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata, epithelial dysplasia, faciodigitogenital dysplasia, familial fibrous dysplasia of jaws, familial white folded dysplasia, fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous dysplasia, hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic dysplasia, mammary dysplasia, mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia, monostotic fibrous dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia, oculoauriculovertebral dysplasia, oculodentodigital dysplasia, oculovertebral dysplasia, odontogenic dysplasia, ophthalmomandibulomelic dysplasia, periapical cemental dysplasia, polyostotic fibrous dysplasia, pseudoachondroplastic spondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia, spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

[0888] Additional pre-neoplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.

[0889] In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognose disorders associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1A, column 8 (Tissue Distribution Library Code).

[0890] In another embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat cancers and neoplasms, including, but not limited to those described herein. In a further preferred embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat acute myelogenous leukemia.

[0891] Additionally, polynucleotides, polypeptides, and/or agonists or antagonists of the invention may affect apoptosis, and therefore, would be useful in treating a number of diseases associated with increased cell survival or the inhibition of apoptosis. For example, diseases associated with increased cell survival or the inhibition of apoptosis that could be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection.

[0892] In preferred embodiments, polynucleotides, polypeptides, and/or agonists or antagonists of the invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.

[0893] Additional diseases or conditions associated with increased cell survival that could be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

[0894] Diseases associated with increased apoptosis that could be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.

[0895] Hyperproliferative diseases and/or disorders that could be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include, but are not limited to, neoplasms located in the liver, abdomen, bone, breast, digestive system, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.

[0896] Similarly, other hyperproliferative disorders can also be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.

[0897] Another preferred embodiment utilizes polynucleotides of the present invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.

[0898] Thus, the present invention provides a method for treating cell proliferative disorders by inserting into an abnormally proliferating cell a polynucleotide of the present invention, wherein said polynucleotide represses said expression.

[0899] Another embodiment of the present invention provides a method of treating cell-proliferative disorders in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells. In a preferred embodiment, polynucleotides of the present invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides. In another preferred embodiment of the present invention, the DNA construct encoding the poynucleotides of the present invention is inserted into cells to be treated utilizing a retrovirus, or more preferably an adenoviral vector (See G J. Nabel, et. al., PNAS 1999 96: 324-326, which is hereby incorporated by reference). In a most preferred embodiment, the viral vector is defective and will not transform non-proliferating cells, only proliferating cells. Moreover, in a preferred embodiment, the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides, can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product. As such the beneficial therapeutic affect of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus.

[0900] Polynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens. By “repressing expression of the oncogenic genes” is intended the suppression of the transcription of the gene, the degradation of the gene transcript (pre-message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.

[0901] For local administration to abnormally proliferating cells, polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification. The polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art. These references are exemplary only and are hereby incorporated by reference. In order to specifically deliver or transfect cells which are abnormally proliferating and spare non-dividing cells, it is preferable to utilize a retrovirus, or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus will be unable to self replicate due to the lack of the retrovirus genes needed for its life cycle. Utilizing such a retroviral delivery system for polynucleotides of the present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.

[0902] The polynucleotides of the present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site. The polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.

[0903] By “cell proliferative disease” is meant any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.

[0904] Any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one of the polynucleotide of the present invention simultaneously to the same site. By “biologically inhibiting” is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells. The biologically inhibitory dose may be determined by assessing the effects of the polynucleotides of the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell cultures, or any other method known to one of ordinary skill in the art.

[0905] The present invention is further directed to antibody-based therapies which involve administering of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, preferably human, patient for treating one or more of the described disorders. Methods for producing anti-polypeptides and anti-polynucleotide antibodies polyclonal and monoclonal antibodies are described in detail elsewhere herein. Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

[0906] A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

[0907] In particular, the antibodies, fragments and derivatives of the present invention are useful for treating a subject having or developing cell proliferative and/or differentiation disorders as described herein. Such treatment comprises administering a single or multiple doses of the antibody, or a fragment, derivative, or a conjugate thereof.

[0908] The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors, for example., which serve to increase the number or activity of effector cells which interact with the antibodies.

[0909] It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragements thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides, including fragements thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, and 10⁻¹⁵M.

[0910] Moreover, polypeptides of the present invention are useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein. In a most preferred embodiment, said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor-specific cells, such as tumor-associated macrophages (See Joseph I B, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated by reference). Antibodies directed to polypeptides or polynucleotides of the present invention may also result in inhibition of angiogenesis directly, or indirectly (See Witte L, et al., Cancer Metastasis Rev. 17(2):155-61 (1998), which is hereby incorporated by reference)).

[0911] Polypeptides, including protein fusions, of the present invention, or fragments thereof may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis. Said polypeptides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death-domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et. al., Eur J Biochem 254(3):439-59 (1998), which is hereby incorporated by reference). Moreover, in another preferred embodiment of the present invention, said polypeptides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of said proteins, either alone or in combination with small molecule drugs or adjuviants, such as apoptonin, galectins, thioredoxins, anti-inflammatory proteins (See for example, Mutat Res 400(1-2):447-55 (1998), Med Hypotheses.50(5):423-33 (1998), Chem Biol Interact. April 24; 111-112:23-34 (1998), J Mol Med.76(6):402-12 (1998), Int J Tissue React;20(1):3-15 (1998), which are all hereby incorporated by reference).

[0912] Polypeptides, including protein fusions to, or fragments thereof, of the present invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result of administering polypeptides, or antibodies directed to said polypeptides as described elsewere herein, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, which is hereby incorporated by reference). Such thereapeutic affects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.

[0913] In another embodiment, the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing polypeptides or polypeptide antibodes associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide of the present invention. Polypeptides or polypeptide antibodes of the invention may be associated with with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.

[0914] Polypeptides, protein fusions to, or fragments thereof, of the present invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the polypeptides of the present invention ‘vaccinated’ the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.

[0915] Renal Disorders

[0916] Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention, may be used to treat, prevent, diagnose, and/or prognose disorders of the renal system. Renal disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention include, but are not limited to, kidney failure, nephritis, blood vessel disorders of kidney, metabolic and congenital kidney disorders, urinary disorders of the kidney, autoimmune disorders, sclerosis and necrosis, electrolyte imbalance, and kidney cancers.

[0917] Kidney diseases which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention include, but are not limited to, acute kidney failure, chronic kidney failure, atheroembolic renal failure, end-stage renal disease, inflammatory diseases of the kidney (e.g., acute glomerulonephritis, postinfectious glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis, familial nephrotic syndrome, membranoproliferative glomerulonephritis I and II, mesangial proliferative glomerulonephritis, chronic glomerulonephritis, acute tubulointerstitial nephritis, chronic tubulointerstitial nephritis, acute post-streptococcal glomerulonephritis (PSGN), pyelonephritis, lupus nephritis, chronic nephritis, interstitial nephritis, and post-streptococcal glomerulonephritis), blood vessel disorders of the kidneys (e.g., kidney infarction, atheroembolic kidney disease, cortical necrosis, malignant nephrosclerosis, renal vein thrombosis, renal underperfusion, renal retinopathy, renal ischemia-reperfusion, renal artery embolism, and renal artery stenosis), and kidney disorders resulting form urinary tract disease (e.g., pyelonephritis, hydronephrosis, urolithiasis (renal lithiasis, nephrolithiasis), reflux nephropathy, urinary tract infections, urinary retention, and acute or chronic unilateral obstructive uropathy.)

[0918] In addition, compositions of the invention can be used to diagnose, prognose, prevent, and/or treat metabolic and congenital disorders of the kidney (e.g., uremia, renal amyloidosis, renal osteodystrophy, renal tubular acidosis, renal glycosuria, nephrogenic diabetes insipidus, cystinuria, Fanconi's syndrome, renal fibrocystic osteosis (renal rickets), Hartnup disease, Bartter's syndrome, Liddle's syndrome, polycystic kidney disease, medullary cystic disease, medullary sponge kidney, Alport's syndrome, nail-patella syndrome, congenital nephrotic syndrome, CRUSH syndrome, horseshoe kidney, diabetic nephropathy, nephrogenic diabetes insipidus, analgesic nephropathy, kidney stones, and membranous nephropathy), and autoimmune disorders of the kidney (e.g., systemic lupus erythematosus (SLE), Goodpasture syndrome, IgA nephropathy, and IgM mesangial proliferative glomerulonephritis).

[0919] Compositions of the invention can also be used to diagnose, prognose, prevent, and/or treat sclerotic or necrotic disorders of the kidney (e.g., glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), necrotizing glomerulonephritis, and renal papillary necrosis), cancers of the kidney (e.g., nephroma, hypemephroma, nephroblastoma, renal cell cancer, transitional cell cancer, renal adenocarcinoma, squamous cell cancer, and Wilm's tumor), and electrolyte imbalances (e.g., nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria, hyponatremia, hypematremia, hypokalemia, hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, and hyperphosphatemia).

[0920] Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Polypeptides may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides are described in more detail herein.

[0921] Cardiovascular Disorders

[0922] Polynucleotides or polypeptides, or agonists or antagonists of the present invention, may be used to treat, prevent, diagnose, and/or prognose cardiovascular disorders, including, but not limited to, peripheral artery disease, such as limb ischemia.

[0923] Cardiovascular disorders include, but are not limited to, cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include, but are not limited to, aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.

[0924] Cardiovascular disorders also include, but are not limited to, heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

[0925] Arrhythmias include, but are not limited to, sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

[0926] Heart valve diseases include, but are not limited to, aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.

[0927] Myocardial diseases include, but are not limited to, alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.

[0928] Myocardial ischemias include, but are not limited to, coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.

[0929] Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

[0930] Aneurysms include, but are not limited to, dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.

[0931] Arterial occlusive diseases include, but are not limited to, arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.

[0932] Cerebrovascular disorders include, but are not limited to, carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.

[0933] Embolisms include, but are not limited to, air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms. Thrombosis include, but are not limited to, coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.

[0934] Ischemic disorders include, but are not limited to, cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis includes, but is not limited to, aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.

[0935] Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Polypeptides may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides are described in more detail herein.

[0936] Respiratory Disorders

[0937] Polynucleotides or polypeptides, or agonists or antagonists of the present invention may be used to treat, prevent, diagnose, and/or prognose diseases and/or disorders of the respiratory system.

[0938] Diseases and disorders of the respiratory system include, but are not limited to, nasal vestibulitis, nonallergic rhinitis (e.g., acute rhinitis, chronic rhinitis, atrophic rhinitis, vasomotor rhinitis), nasal polyps, and sinusitis, juvenile angiofibromas, cancer of the nose and juvenile papillomas, vocal cord polyps, nodules (singer's nodules), contact ulcers, vocal cord paralysis, laryngoceles, pharyngitis (e.g., viral and bacterial), tonsillitis, tonsillar cellulitis, parapharyngeal abscess, laryngitis, laryngoceles, and throat cancers (e.g., cancer of the nasopharynx, tonsil cancer, larynx cancer), lung cancer (e.g., squamous cell carcinoma, small cell (oat cell) carcinoma, large cell carcinoma, and adenocarcinoma), allergic disorders (eosinophilic pneumonia, hypersensitivity pneumonitis (e.g., extrinsic allergic alveolitis, allergic interstitial pneumonitis, organic dust pneumoconiosis, allergic bronchopulmonary aspergillosis, asthma, Wegener's granulomatosis (granulomatous vasculitis), Goodpasture's syndrome)), pneumonia (e.g., bacterial pneumonia (e.g., Streptococcus pneumoniae (pneumoncoccal pneumonia), Staphylococcus aureus (staphylococcal pneumonia), Gram-negative bacterial pneumonia (caused by, e.g., Klebsiella and Pseudomas spp.), Mycoplasma pneumoniae pneumonia, Hemophilus influenzae pneumonia, Legionella pneumophila (Legionnaires' disease), and Chlamydia psittaci (Psittacosis)), and viral pneumonia (e.g., influenza, chickenpox (varicella).

[0939] Additional diseases and disorders of the respiratory system include, but are not limited to bronchiolitis, polio (poliomyelitis), croup, respiratory syncytial viral infection, mumps, erythema infectiosum (fifth disease), roseola infantum, progressive rubella panencephalitis, german measles, and subacute sclerosing panencephalitis), fungal pneumonia (e.g., Histoplasmosis, Coccidioidomycosis, Blastomycosis, fungal infections in people with severely suppressed immune systems (e.g., cryptococcosis, caused by Cryptococcus neoformans; aspergillosis, caused by Aspergillus spp.; candidiasis, caused by Candida; and mucormycosis)), Pneumocystis carinii (pneumocystis pneumonia), atypical pneumonias (e.g., Mycoplasma and Chlamydia spp.), opportunistic infection pneumonia, nosocomial pneumonia, chemical pneumonitis, and aspiration pneumonia, pleural disorders (e.g., pleurisy, pleural effusion, and pneumothorax (e.g., simple spontaneous pneumothorax, complicated spontaneous pneumothorax, tension pneumothorax)), obstructive airway diseases (e.g., asthma, chronic obstructive pulmonary disease (COPD), emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis, black lung (coal workers' pneumoconiosis), asbestosis, berylliosis, occupational asthsma, byssinosis, and benign pneumoconioses), Infiltrative Lung Disease (e.g., pulmonary fibrosis (e.g., fibrosing alveolitis, usual interstitial pneumonia), idiopathic pulmonary fibrosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, histiocytosis X (e.g., Letterer-Siwe disease, Hand-Schüller-Christian disease, eosinophilic granuloma), idiopathic pulmonary hemosiderosis, sarcoidosis and pulmonary alveolar proteinosis), Acute respiratory distress syndrome (also called, e.g., adult respiratory distress syndrome), edema, pulmonary embolism, bronchitis (e.g., viral, bacterial), bronchiectasis, atelectasis, lung abscess (caused by, e.g., Staphylococcus aureus or Legionella pneumophila), and cystic fibrosis.

[0940] Anti-Angiogenesis Activity

[0941] The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate. Rastinejad et al., Cell 56:345-355 (1989). In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkman et al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science 221:719-725 (1983). In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).

[0942] The present invention provides for treatment of diseases or disorders associated with neovascularization by administration of the polynucleotides and/or polypeptides of the invention, as well as agonists or antagonists of the present invention. Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)). Thus, the present invention provides a method of treating an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist of the invention. For example, polynucleotides, polypeptides, antagonists and/or agonists may be utilized in a variety of additional methods in order to therapeutically treat a cancer or tumor. Cancers which may be treated with polynucleotides, polypeptides, antagonists and/or agonists include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemias. For example, polynucleotides, polypeptides, antagonists and/or agonists may be delivered topically, in order to treat cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.

[0943] Within yet other aspects, polynucleotides, polypeptides, antagonists and/or agonists may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration. Polynucleotides, polypeptides, antagonists and/or agonists may be delivered directly into the tumor, or near the tumor site, via injection or a catheter. Of course, as the artisan of ordinary skill will appreciate, the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.

[0944] Polynucleotides, polypeptides, antagonists and/or agonists may be useful in treating other disorders, besides cancers, which involve angiogenesis. These disorders include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.

[0945] For example, within one aspect of the present invention methods are provided for treating hypertrophic scars and keloids, comprising the step of administering a polynucleotide, polypeptide, antagonist and/or agonist of the invention to a hypertrophic scar or keloid.

[0946] Within one embodiment of the present invention polynucleotides, polypeptides, antagonists and/or agonists of the invention are directly injected into a hypertrophic scar or keloid, in order to prevent the progression of these lesions. This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids (e.g., burns), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development. As noted above, the present invention also provides methods for treating neovascular diseases of the eye, including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.

[0947] Moreover, Ocular disorders associated with neovascularization which can be treated with the polynucleotides and polypeptides of the present invention (including agonists and/or antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312 (1978).

[0948] Thus, within one aspect of the present invention methods are provided for treating neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization), comprising the step of administering to a patient a therapeutically effective amount of a compound (as described above) to the cornea, such that the formation of blood vessels is inhibited. Briefly, the cornea is a tissue which normally lacks blood vessels. In certain pathological conditions however, capillaries may extend into the cornea from the pericorneal vascular plexus of the limbus. When the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates. A wide variety of disorders can result in corneal neovascularization, including for example, corneal infections (e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.

[0949] Within particularly preferred embodiments of the invention, may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form. The solution or suspension may be prepared in its pure form and administered several times daily. Alternatively, anti-angiogenic compositions, prepared as described above, may also be administered directly to the cornea. Within preferred embodiments, the anti-angiogenic composition is prepared with a muco-adhesive polymer which binds to cornea. Within further embodiments, the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy. Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.

[0950] Within other embodiments, the compounds described above may be injected directly into the corneal stroma by an ophthalmologist under microscopic guidance. The preferred site of injection may vary with the morphology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic corneal injection to “protect” the cornea from the advancing blood vessels. This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization. In this situation the material could be injected in the perilimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply. Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas. In a sustained-release form injections might only be required 2-3 times per year. A steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself.

[0951] Within another aspect of the present invention, methods are provided for treating neovascular glaucoma, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. In one embodiment, the compound may be administered topically to the eye in order to treat early forms of neovascular glaucoma. Within other embodiments, the compound may be implanted by injection into the region of the anterior chamber angle. Within other embodiments, the compound may also be placed in any location such that the compound is continuously released into the aqueous humor. Within another aspect of the present invention, methods are provided for treating proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eyes, such that the formation of blood vessels is inhibited.

[0952] Within particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist and/or agonist in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.

[0953] Within another aspect of the present invention, methods are provided for treating retrolental fibroplasia, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. The compound may be administered topically, via intravitreous injection and/or via intraocular implants.

[0954] Additionally, disorders which can be treated with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

[0955] Moreover, disorders and/or states, which can be treated, prevented, diagnosed, and/or prognosed with the the polynucleotides, polypeptides, agonists and/or agonists of the invention include, but are not limited to, solid tumors, blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control agent by preventing vascularization required for embryo implantation controlling menstruation, diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

[0956] In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a “morning after” method. Polynucleotides, polypeptides, agonists and/or agonists may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.

[0957] Polynucleotides, polypeptides, agonists and/or agonists of the present invention may be incorporated into surgical sutures in order to prevent stitch granulomas.

[0958] Polynucleotides, polypeptides, agonists and/or agonists may be utilized in a wide variety of surgical procedures. For example, within one aspect of the present invention a compositions (in the form of, for example, a spray or film) may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues. Within other aspects of the present invention, compositions (e.g., in the form of a spray) may be delivered via endoscopic procedures in order to coat tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects of the present invention, surgical meshes which have been coated with anti-angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical mesh might be utilized. For example, within one embodiment of the invention a surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.

[0959] Within further aspects of the present invention, methods are provided for treating tumor excision sites, comprising administering a polynucleotide, polypeptide, agonist and/or agonist to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited. Within one embodiment of the invention, the anti-angiogenic compound is administered directly to the tumor excision site (e.g., applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration. Within particularly preferred embodiments of the invention, the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.

[0960] Within one aspect of the present invention, polynucleotides, polypeptides, agonists and/or agonists may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors. For example, within one embodiment of the invention, anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.

[0961] The polynucleotides, polypeptides, agonists and/or agonists of the present invention may also be administered along with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: Anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.

[0962] Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.

[0963] Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.

[0964] Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.

[0965] A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminolmidazole; and metalloproteinase inhibitors such as BB94.

[0966] Diseases at the Cellular Level

[0967] Diseases associated with increased cell survival or the inhibition of apoptosis that could be treated, prevented, diagnosed, and/or prognosed using polynucleotides or polypeptides, as well as antagonists or agonists of the present invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection.

[0968] In preferred embodiments, polynucleotides, polypeptides, and/or antagonists of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above.

[0969] Additional diseases or conditions associated with increased cell survival that could be treated or detected by polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

[0970] Diseases associated with increased apoptosis that could be treated, prevented, diagnosed, and/or prognesed using polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, include, but are not limited to, AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.

[0971] Wound Healing and Epithelial Cell Proliferation

[0972] In accordance with yet a further aspect of the present invention, there is provided a process for utilizing polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and complications associated with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to promote dermal reestablishment subsequent to dermal loss

[0973] Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are types of grafts that polynucleotides or polypeptides, agonists or antagonists of the present invention, could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermic graft, autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, can be used to promote skin strength and to improve the appearance of aged skin.

[0974] It is believed that polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. Polynucleotides or polypeptides, agonists or antagonists of the present invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

[0975] Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may have a cytoprotective effect on the small intestine mucosa. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.

[0976] Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with polynucleotides or polypeptides, agonists or antagonists of the present invention, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to treat diseases associate with the under expression.

[0977] Moreover, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to prevent and heal damage to the lungs due to various pathological states. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated using polynucleotides or polypeptides, agonists or antagonists of the present invention. Also, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants.

[0978] Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).

[0979] In addition, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Types I and II diabetes, where some islet cell function remains, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.

[0980] Neural Activity and Neurological Diseases

[0981] The polynucleotides, polypeptides and agonists or antagonists of the invention may be used for the diagnosis and/or treatment of diseases, disorders, damage or injury of the brain and/or nervous system. Nervous system disorders that can be treated with the compositions of the invention (e.g., polypeptides, polynucleotides, and/or agonists or antagonists), include, but are not limited to, nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the methods of the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, or syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to, degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including, but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

[0982] In one embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to protect neural cells from the damaging effects of hypoxia. In a further preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to protect neural cells from the damaging effects of cerebral hypoxia. According to this embodiment, the compositions of the invention are used to treat or prevent neural cell injury associated with cerebral hypoxia. In one non-exclusive aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention, are used to treat or prevent neural cell injury associated with cerebral ischemia. In another non-exclusive aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with cerebral infarction.

[0983] In another preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with a stroke. In a specific embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent cerebral neural cell injury associated with a stroke.

[0984] In another preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with a heart attack. In a specific embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent cerebral neural cell injury associated with a heart attack.

[0985] The compositions of the invention which are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, compositions of the invention which elicit any of the following effects may be useful according to the invention: (1) increased survival time of neurons in culture either in the presence or absence of hypoxia or hypoxic conditions; (2) increased sprouting of neurons in culture or in vivo; (3) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms of neuron dysfunction in vivo. Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, in Zhang et al., Proc Natl Acad Sci USA 97:3637-42 (2000) or in Arakawa et al., J. Neurosci., 10:3507-15 (1990); increased sprouting of neurons may be detected by methods known in the art, such as, for example, the methods set forth in Pestronk et al., Exp. Neurol., 70:65-82 (1980), or Brown et al., Ann. Rev. Neurosci., 4:17-42 (1981); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., using techniques known in the art and depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.

[0986] In specific embodiments, motor neuron disorders that may be treated according to the invention include, but are not limited to, disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

[0987] Further, polypeptides or polynucleotides of the invention may play a role in neuronal survival; synapse formation; conductance; neural differentiation, etc. Thus, compositions of the invention (including polynucleotides, polypeptides, and agonists or antagonists) may be used to diagnose and/or treat or prevent diseases or disorders associated with these roles, including, but not limited to, learning and/or cognition disorders. The compositions of the invention may also be useful in the treatment or prevention of neurodegenerative disease states and/or behavioural disorders. Such neurodegenerative disease states and/or behavioral disorders include, but are not limited to, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, compositions of the invention may also play a role in the treatment, prevention and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders.

[0988] Additionally, polypeptides, polynucleotides and/or agonists or antagonists of the invention, may be useful in protecting neural cells from diseases, damage, disorders, or injury, associated with cerebrovascular disorders including, but not limited to, carotid artery diseases (e.g., carotid artery thrombosis, carotid stenosis, or Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis (e.g., carotid artery thrombosis, sinus thrombosis, or Wallenberg's Syndrome), cerebral hemorrhage (e.g., epidural or subdural hematoma, or subarachnoid hemorrhage), cerebral infarction, cerebral ischemia (e.g., transient cerebral ischemia, Subclavian Steal Syndrome, or vertebrobasilar insufficiency), vascular dementia (e.g., multi-infarct), leukomalacia, periventricular, and vascular headache (e.g., cluster headache or migraines).

[0989] In accordance with yet a further aspect of the present invention, there is provided a process for utilizing polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, for therapeutic purposes, for example, to stimulate neurological cell proliferation and/or differentiation. Therefore, polynucleotides, polypeptides, agonists and/or antagonists of the invention may be used to treat and/or detect neurologic diseases. Moreover, polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used as a marker or detector of a particular nervous system disease or disorder.

[0990] Examples of neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include brain diseases, such as metabolic brain diseases which includes phenylketonuria such as maternal phenylketonuria, pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, Wernicke's Encephalopathy, brain edema, brain neoplasms such as cerebellar neoplasms which include infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms, supratentorial neoplasms, canavan disease, cerebellar diseases such as cerebellar ataxia which include spinocerebellar degeneration such as ataxia telangiectasia, cerebellar dyssynergia, Friederich's Ataxia, Machado-Joseph Disease, olivopontocerebellar atrophy, cerebellar neoplasms such as infratentorial neoplasms, diffuse cerebral sclerosis such as encephalitis periaxialis, globoid cell leukodystrophy, metachromatic leukodystrophy and subacute sclerosing panencephalitis.

[0991] Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include cerebrovascular disorders (such as carotid artery diseases which include carotid artery thrombosis, carotid stenosis and Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis such as carotid artery thrombosis, sinus thrombosis and Wallenberg's Syndrome, cerebral hemorrhage such as epidural hematoma, subdural hematoma and subarachnoid hemorrhage, cerebral infarction, cerebral ischemia such as transient cerebral ischemia, Subclavian Steal Syndrome and vertebrobasilar insufficiency, vascular dementia such as multi-infarct dementia, periventricular leukomalacia, vascular headache such as cluster headache and migraine.

[0992] Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include dementia such as AIDS Dementia Complex, presenile dementia such as Alzheimer's Disease and Creutzfeldt-Jakob Syndrome, senile dementia such as Alzheimer's Disease and progressive supranuclear palsy, vascular dementia such as multi-infarct dementia, encephalitis which include encephalitis periaxialis, viral encephalitis such as epidemic encephalitis, Japanese Encephalitis, St. Louis Encephalitis, tick-borne encephalitis and West Nile Fever, acute disseminated encephalomyelitis, meningoencephalitis such as uveomeningoencephalitic syndrome, Postencephalitic Parkinson Disease and subacute sclerosing panencephalitis, encephalomalacia such as periventricular leukomalacia, epilepsy such as generalized epilepsy which includes infantile spasms, absence epilepsy, myoclonic epilepsy which includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsy such as complex partial epilepsy, frontal lobe epilepsy and temporal lobe epilepsy, post-traumatic epilepsy, status epilepticus such as Epilepsia Partialis Continua, and Hallervorden-Spatz Syndrome.

[0993] Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include hydrocephalus such as Dandy-Walker Syndrome and normal pressure hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria, narcolepsy which includes cataplexy, bulbar poliomyelitis, cerebri pseudotumor, Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebral toxoplasmosis, intracranial tuberculoma and Zellweger Syndrome, central nervous system infections such as AIDS Dementia Complex, Brain Abscess, subdural empyema, encephalomyelitis such as Equine Encephalomyelitis, Venezuelan Equine Encephalomyelitis, Necrotizing Hemorrhagic Encephalomyelitis, Visna, and cerebral malaria.

[0994] Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include meningitis such as arachnoiditis, aseptic meningtitis such as viral meningtitis which includes lymphocytic choriomeningitis, Bacterial meningtitis which includes Haemophilus Meningtitis, Listeria Meningtitis, Meningococcal Meningtitis such as Waterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and meningeal tuberculosis, fungal meningitis such as Cryptococcal Meningtitis, subdural effusion, meningoencephalitis such as uvemeningoencephalitic syndrome, myelitis such as transverse myelitis, neurosyphilis such as tabes dorsalis, poliomyelitis which includes bulbar poliomyelitis and postpoliomyelitis syndrome, prion diseases (such as Creutzfeldt-Jakob Syndrome, Bovine Spongiform Encephalopathy, Gerstmann-Straussler Syndrome, Kuru, Scrapie), and cerebral toxoplasmosis.

[0995] Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include central nervous system neoplasms such as brain neoplasms that include cerebellar neoplasms such as infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms and supratentorial neoplasms, meningeal neoplasms, spinal cord neoplasms which include epidural neoplasms, demyelinating diseases such as Canavan Diseases, diffuse cerebral sceloris which includes adrenoleukodystrophy, encephalitis periaxialis, globoid cell leukodystrophy, diffuse cerebral sclerosis such as metachromatic leukodystrophy, allergic encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, progressive multifocal leukoencephalopathy, multiple sclerosis, central pontine myelinolysis, transverse myelitis, neuromyelitis optica, Scrapie, Swayback, Chronic Fatigue Syndrome, Visna, High Pressure Nervous Syndrome, Meningism, spinal cord diseases such as amyotonia congenita, amyotrophic lateral sclerosis, spinal muscular atrophy such as Werdnig-Hoffmann Disease, spinal cord compression, spinal cord neoplasms such as epidural neoplasms, syringomyelia, Tabes Dorsalis, Stiff-Man Syndrome, mental retardation such as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's Syndrome, Down Syndrome, Gangliosidoses such as gangliosidoses G(M1), Sandhoff Disease, Tay-Sachs Disease, Hartnup Disease, homocystinuria, Laurence-Moon-Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup Urine Disease, mucolipidosis such as fucosidosis, neuronal ceroid-lipofuscinosis, oculocerebrorenal syndrome, phenylketonuria such as maternal phenylketonuria, Prader-Willi Syndrome, Rett Syndrome, Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR Syndrome, nervous system abnormalities such as holoprosencephaly, neural tube defects such as anencephaly which includes hydrangencephaly, Arnold-Chairi Deformity, encephalocele, meningocele, meningomyelocele, spinal dysraphism such as spina bifida cystica and spina bifida occulta.

[0996] Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include hereditary motor and sensory neuropathies which include Charcot-Marie Disease, Hereditary optic atrophy, Refsum's Disease, hereditary spastic paraplegia, Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies such as Congenital Analgesia and Familial Dysautonomia, Neurologic manifestations (such as agnosia that include Gerstmann's Syndrome, Amnesia such as retrograde amnesia, apraxia, neurogenic bladder, cataplexy, communicative disorders such as hearing disorders that includes deafness, partial hearing loss, loudness recruitment and tinnitus, language disorders such as aphasia which include agraphia, anomia, broca aphasia, and Wernicke Aphasia, Dyslexia such as Acquired Dyslexia, language development disorders, speech disorders such as aphasia which includes anomia, broca aphasia and Wernicke Aphasia, articulation disorders, communicative disorders such as speech disorders which include dysarthria, echolalia, mutism and stuttering, voice disorders such as aphonia and hoarseness, decerebrate state, delirium, fasciculation, hallucinations, meningism, movement disorders such as angelman syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia, muscle hypotonia, myoclonus, tic, torticollis and tremor, muscle hypertonia such as muscle rigidity such as stiff-man syndrome, muscle spasticity, paralysis such as facial paralysis which includes Herpes Zoster Oticus, Gastroparesis, Hemiplegia, ophthalmoplegia such as diplopia, Duane's Syndrome, Horner's Syndrome, Chronic progressive external ophthalmoplegia such as Kearns Syndrome, Bulbar Paralysis, Tropical Spastic Paraparesis, Paraplegia such as Brown-Sequard Syndrome, quadriplegia, respiratory paralysis and vocal cord paralysis, paresis, phantom limb, taste disorders such as ageusia and dysgeusia, vision disorders such as amblyopia, blindness, color vision defects, diplopia, hemianopsia, scotoma and subnormal vision, sleep disorders such as hypersomnia which includes Kleine-Levin Syndrome, insomnia, and somnambulism, spasm such as trismus, unconsciousness such as coma, persistent vegetative state and syncope and vertigo, neuromuscular diseases such as amyotonia congenita, amyotrophic lateral sclerosis, Lambert-Eaton Myasthenic Syndrome, motor neuron disease, muscular atrophy such as spinal muscular atrophy, Charcot-Marie Disease and Werdnig-Hoffmann Disease, Postpoliomyelitis Syndrome, Muscular Dystrophy, Myasthenia Gravis, Myotonia Atrophica, Myotonia Confenita, Nemaline Myopathy, Familial Periodic Paralysis, Multiplex Paramyloclonus, Tropical Spastic Paraparesis and Stiff-Man Syndrome, peripheral nervous system diseases such as acrodynia, amyloid neuropathies, autonomic nervous system diseases such as Adie's Syndrome, Barre-Lieou Syndrome, Familial Dysautonomia, Homer's Syndrome, Reflex Sympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseases such as Acoustic Nerve Diseases such as Acoustic Neuroma which includes Neurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia, Melkersson-Rosenthal Syndrome, ocular motility disorders which includes amblyopia, nystagmus, oculomotor nerve paralysis, ophthalmoplegia such as Duane's Syndrome, Homer's Syndrome, Chronic Progressive External Ophthalmoplegia which includes Kearns Syndrome, Strabismus such as Esotropia and Exotropia, Oculomotor Nerve Paralysis, Optic Nerve Diseases such as Optic Atrophy which includes Hereditary Optic Atrophy, Optic Disk Drusen, Optic Neuritis such as Neuromyelitis Optica, Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis, Demyelinating Diseases such as Neuromyelitis Optica-and Swayback, and Diabetic neuropathies such as diabetic foot.

[0997] Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include nerve compression syndromes such as carpal tunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve compression syndrome, neuralgia such as causalgia, cervico-brachial neuralgia, facial neuralgia and trigeminal neuralgia, neuritis such as experimental allergic neuritis, optic neuritis, polyneuritis, polyradiculoneuritis and radiculities such as polyradiculitis, hereditary motor and sensory neuropathies such as Charcot-Marie Disease, Hereditary Optic Atrophy, Refsum's Disease, Hereditary Spastic Paraplegia and Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies which include Congenital Analgesia and Familial Dysautonomia, POEMS Syndrome, Sciatica, Gustatory Sweating and Tetany).

[0998] Endocrine Disorders

[0999] Polynucleotides or polypeptides, or agonists or antagonists of the present invention, may be used to treat, prevent, diagnose, and/or prognose disorders and/or diseases related to hormone imbalance, and/or disorders or diseases of the endocrine system.

[1000] Hormones secreted by the glands of the endocrine system control physical growth, sexual function, metabolism, and other functions. Disorders may be classified in two ways: disturbances in the production of hormones, and the inability of tissues to respond to hormones. The etiology of these hormone imbalance or endocrine system diseases, disorders or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy, injury or toxins), or infectious. Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention can be used as a marker or detector of a particular disease or disorder related to the endocrine system and/or hormone imbalance.

[1001] Endocrine system and/or hormone imbalance and/or diseases encompass disorders of uterine motility including, but not limited to: complications with pregnancy and labor (e.g., pre-term labor, post-term pregnancy, spontaneous abortion, and slow or stopped labor); and disorders and/or diseases of the menstrual cycle (e.g., dysmenorrhea and endometriosis).

[1002] Endocrine system and/or hormone imbalance disorders and/or diseases include disorders and/or diseases of the pancreas, such as, for example, diabetes mellitus, diabetes insipidus, congenital pancreatic agenesis, pheochromocytoma—islet cell tumor syndrome; disorders and/or diseases of the adrenal glands such as, for example, Addison's Disease, corticosteroid deficiency, virilizing disease, hirsutism, Cushing's Syndrome, hyperaldosteronism, pheochromocytoma; disorders and/or diseases of the pituitary gland, such as, for example, hyperpituitarism, hypopituitarism, pituitary dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, gigantism; disorders and/or diseases of the thyroid, including but not limited to, hyperthyroidism, hypothyroidism, Plummer's disease, Graves' disease (toxic diffuse goiter), toxic nodular goiter, thyroiditis (Hashimoto's thyroiditis, subacute granulomatous thyroiditis, and silent lymphocytic thyroiditis), Pendred's syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormone coupling defect, thymic aplasia, Hurthle cell tumours of the thyroid, thyroid cancer, thyroid carcinoma, Medullary thyroid carcinoma; disorders and/or diseases of the parathyroid, such as, for example, hyperparathyroidism, hypoparathyroidism; disorders and/or diseases of the hypothalamus.

[1003] In specific embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides, agonists and antagonists, may be used to diagnose, prognose, treat, prevent, or ameliorate diseases and disorders associated with aberrant glucose metabolism or glucose uptake into cells.

[1004] In a specific embodiment, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists and/or antagonists thereof may be used to diagnose, prognose, treat, prevent, and/or ameliorate type I diabetes mellitus (insulin dependent diabetes mellitus, IDDM).

[1005] In another embodiment, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists and/or antagonists thereof may be used to diagnose, prognose, treat, prevent, and/or ameliorate type II diabetes mellitus (insulin resistant diabetes mellitus).

[1006] Additionally, in other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or antagonists thereof (especially neutralizing or antagonistic antibodies) may be used to diagnose, prognose, treat, prevent, and/or ameliorate conditions associated with (type I or type II) diabetes mellitus, including, but not limited to, diabetic ketoacidosis, diabetic coma, nonketotic hyperglycemic-hyperosmolar coma, seizures, mental confusion, drowsiness, cardiovascular disease (e.g., heart disease, atherosclerosis, microvascular disease, hypertension, stroke, and other diseases and disorders as described in the “Cardiovascular Disorders” section), dyslipidemia, kidney disease (e.g., renal failure, nephropathy other diseases and disorders as described in the “Renal Disorders” section), nerve damage, neuropathy, vision impairment (e.g., diabetic retinopathy and blindness), ulcers and impaired wound healing, infections (e.g., infectious diseases and disorders as described in the “Infectious Diseases” section, especially of the urinary tract and skin), carpal tunnel syndrome and Dupuytren's contracture.

[1007] In other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to regulate the animal's weight. In specific embodiments the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to control the animal's weight by modulating a biochemical pathway involving insulin. In still other embodiments the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to control the animal's weight by modulating a biochemical pathway involving insulin-like growth factor.

[1008] In addition, endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases of the testes or ovaries, including cancer. Other disorders and/or diseases of the testes or ovaries further include, for example, ovarian cancer, polycystic ovary syndrome, Klinefelter's syndrome, vanishing testes syndrome (bilateral anorchia), congenital absence of Leydig's cells, cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillary haemangioma of the testis (benign), neoplasias of the testis and neo-testis.

[1009] Moreover, endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases such as, for example, polyglandular deficiency syndromes, pheochromocytoma, neuroblastoma, multiple Endocrine neoplasia, and disorders and/or cancers of endocrine tissues.

[1010] In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose, prognose, prevent, and/or treat endocrine diseases and/or disorders associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1A, column 8 (Tissue Distribution Library Code).

[1011] Reproductive System Disorders

[1012] The polynucleotides or polypeptides, or agonists or antagonists of the invention may be used for the diagnosis, treatment, or prevention of diseases and/or disorders of the reproductive system. Reproductive system disorders that can be treated by the compositions of the invention, include, but are not limited to, reproductive system injuries, infections, neoplastic disorders, congenital defects, and diseases or disorders which result in infertility, complications with pregnancy, labor, or parturition, and postpartum difficulties.

[1013] Reproductive system disorders and/or diseases include diseases and/or disorders of the testes, including testicular atrophy, testicular feminization, cryptorchism (unilateral and bilateral), anorchia, ectopic testis, epididymitis and orchitis (typically resulting from infections such as, for example, gonorrhea, mumps, tuberculosis, and syphilis), testicular torsion, vasitis nodosa, germ cell tumors (e.g., seminomas, embryonal cell carcinomas, teratocarcinomas, choriocarcinomas, yolk sac tumors, and teratomas), stromal tumors (e.g., Leydig cell tumors), hydrocele, hematocele, varicocele, spermatocele, inguinal hernia, and disorders of sperm production (e.g., immotile cilia syndrome, aspermia, asthenozoospermia, azoospermia, oligospermia, and teratozoospermia).

[1014] Reproductive system disorders also include disorders of the prostate gland, such as acute non-bacterial prostatitis, chronic non-bacterial prostatitis, acute bacterial prostatitis, chronic bacterial prostatitis, prostatodystonia, prostatosis, granulomatous prostatitis, malacoplakia, benign prostatic hypertrophy or hyperplasia, and prostate neoplastic disorders, including adenocarcinomas, transitional cell carcinomas, ductal carcinomas, and squamous cell carcinomas.

[1015] Additionally, the compositions of the invention may be useful in the diagnosis, treatment, and/or prevention of disorders or diseases of the penis and urethra, including inflammatory disorders, such as balanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis, syphilis, herpes simplex virus, gonorrhea, non-gonococcal urethritis, chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome, condyloma acuminatum, condyloma latum, and pearly penile papules; urethral abnormalities, such as hypospadias, epispadias, and phimosis; premalignant lesions, including Erythroplasia of Queyrat, Bowen's disease, Bowenoid paplosis, giant condyloma of Buscke-Lowenstein, and varrucous carcinoma; penile cancers, including squamous cell carcinomas, carcinoma in situ, verrucous carcinoma, and disseminated penile carcinoma; urethral neoplastic disorders, including penile urethral carcinoma, bulbomembranous urethral carcinoma, and prostatic urethral carcinoma; and erectile disorders, such as priapism, Peyronie's disease, erectile dysfunction, and impotence.

[1016] Moreover, diseases and/or disorders of the vas deferens include vasculititis and CBAVD (congenital bilateral absence of the vas deferens); additionally, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the diagnosis, treatment, and/or prevention of diseases and/or disorders of the seminal vesicles, including hydatid disease, congenital chloride diarrhea, and polycystic kidney disease.

[1017] Other disorders and/or diseases of the male reproductive system include, for example, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, high fever, multiple sclerosis, and gynecomastia.

[1018] Further, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the diagnosis, treatment, and/or prevention of diseases and/or disorders of the vagina and vulva, including bacterial vaginosis, candida vaginitis, herpes simplex virus, chancroid, granuloma inguinale, lymphogranuloma venereum, scabies, human papillomavirus, vaginal trauma, vulvar trauma, adenosis, chlamydia vaginitis, gonorrhea, trichomonas vaginitis, condyloma acuminatum, syphilis, molluscum contagiosum, atrophic vaginitis, Paget's disease, lichen sclerosus, lichen planus, vulvodynia, toxic shock syndrome, vaginismus, vulvovaginitis, vulvar vestibulitis, and neoplastic disorders, such as squamous cell hyperplasia, clear cell carcinoma, basal cell carcinoma, melanomas, cancer of Bartholin's gland, and vulvar intraepithelial neoplasia.

[1019] Disorders and/or diseases of the uterus include dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding (e.g., due to aberrant hormonal signals), and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas, and sarcomas. Additionally, the polypeptides, polynucleotides, or agonists or antagonists of the invention may be useful as a marker or detector of, as well as in the diagnosis, treatment, and/or prevention of congenital uterine abnormalities, such as bicornuate uterus, septate uterus, simple unicornuate uterus, unicornuate uterus with a noncavitary rudimentary horn, unicornuate uterus with a non-communicating cavitary rudimentary horn, unicornuate uterus with a communicating cavitary horn, arcuate uterus, uterine didelfus, and T-shaped uterus.

[1020] Ovarian diseases and/or disorders include anovulation, polycystic ovary syndrome (Stein-Leventhal syndrome), ovarian cysts, ovarian hypofunction, ovarian insensitivity to gonadotropins, ovarian overproduction of androgens, right ovarian vein syndrome, amenorrhea, hirutism, and ovarian cancer (including, but not limited to, primary and secondary cancerous growth, Sertoli-Leydig tumors, endometriod carcinoma of the ovary, ovarian papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma, and Ovarian Krukenberg tumors).

[1021] Cervical diseases and/or disorders include cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, and cervical neoplasms (including, for example, cervical carcinoma, squamous metaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, and columnar cell neoplasia).

[1022] Additionally, diseases and/or disorders of the reproductive system include disorders and/or diseases of pregnancy, including miscarriage and stillbirth, such as early abortion, late abortion, spontaneous abortion, induced abortion, therapeutic abortion, threatened abortion, missed abortion, incomplete abortion, complete abortion, habitual abortion, missed abortion, and septic abortion; ectopic pregnancy, anemia, Rh incompatibility, vaginal bleeding during pregnancy, gestational diabetes, intrauterine growth retardation, polyhydramnios, HELLP syndrome, abruptio placentae, placenta previa, hyperemesis, preeclampsia, eclampsia, herpes gestationis, and urticaria of pregnancy. Additionally, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the diagnosis, treatment, and/or prevention of diseases that can complicate pregnancy, including heart disease, heart failure, rheumatic heart disease, congenital heart disease, mitral valve prolapse, high blood pressure, anemia, kidney disease, infectious disease (e.g., rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydia, HMV, AIDS, and genital herpes), diabetes mellitus, Graves' disease, thyroiditis, hypothyroidism, Hashimoto's thyroiditis, chronic active hepatitis, cirrhosis of the liver, primary biliary cirrhosis, asthma, systemic lupus eryematosis, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis, ovarian cysts, gallbladder disorders,and obstruction of the intestine.

[1023] Complications associated with labor and parturition include premature rupture of the membranes, pre-term labor, post-term pregnancy, postmaturity, labor that progresses too slowly, fetal distress (e.g., abnormal heart rate (fetal or maternal), breathing problems, and abnormal fetal position), shoulder dystocia, prolapsed umbilical cord, amniotic fluid embolism, and aberrant uterine bleeding.

[1024] Further, diseases and/or disorders of the postdelivery period, including endometritis, myometritis, parametritis, peritonitis, pelvic thrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis, saphenous thrombophlebitis, mastitis, cystitis, postpartum hemorrhage, and inverted uterus.

[1025] Other disorders and/or diseases of the female reproductive system that may be diagnosed, treated, and/or prevented by the polynucleotides, polypeptides, and agonists or antagonists of the present invention include, for example, Turner's syndrome, pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory disease, pelvic congestion (vascular engorgement), frigidity, anorgasmia, dyspareunia, ruptured fallopian tube, and Mittelschmerz.

[1026] Infectious Disease

[1027] Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.

[1028] Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention. Examples of viruses, include, but are not limited to Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines. In a further specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat AIDS.

[1029] Similarly, bacterial and fungal agents that can cause disease or symptoms and that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following Gram-Negative and Gram-positive bacteria, bacterial families, and fungi: Actinomyces (e.g., Norcardia), Acinetobacter, Cryptococcus neoformans, Aspergillus, Bacillaceae (e.g., Bacillus anthrasis), Bacteroides (e.g., Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucella, Candidia, Campylobacter, Chlamydia, Clostridium (e.g., Clostridium botulinum, Clostridium dificile, Clostridium perfringens, Clostridium tetani), Coccidioides, Corynebacterium (e.g., Corynebacterium diptheriae), Cryptococcus, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli), Enterobacter (e.g. Enterobacter aerogenes), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis, Salmonella typhi), Serratia, Yersinia, Shigella), Erysipelothrix, Haemophilus (e.g., Haemophilus influenza type B), Helicobacter, Legionella (e.g., Legionella pneumophila), Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio cholerae), Neisseriaceae (e.g., Neisseria gonorrhea, Neisseria meningitidis), Pasteurellacea, Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa), Rickettsiaceae, Spirochetes (e.g., Treponema spp., Leptospira spp., Borrelia spp.), Shigella spp., Staphylococcus (e.g., Staphylococcus aureus), Meningiococcus, Pneumococcus and Streptococcus (e.g., Streptococcus pneumoniae and Groups A, B, and C Streptococci), and Ureaplasmas. These bacterial, parasitic, and fungal families can cause diseases or symptoms, including, but not limited to: antibiotic-resistant infections, bacteremia, endocarditis, septicemia, eye infections (e.g., conjunctivitis), uveitis, tuberculosis, gingivitis, bacterial diarrhea, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, dental caries, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, dysentery, paratyphoid fever, food poisoning, Legionella disease, chronic and acute inflammation, erythema, yeast infections, typhoid, pneumonia, gonorrhea, meningitis (e.g., mengitis types A and B), chlamydia, syphillis, diphtheria, leprosy, brucellosis, peptic ulcers, anthrax, spontaneous abortions, birth defects, pneumonia, lung infections, ear infections, deafness, blindness, lethargy, malaise, vomiting, chronic diarrhea, Crohn's disease, colitis, vaginosis, sterility, pelvic inflammatory diseases, candidiasis, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections, noscomial infections. Polynucleotides or polypeptides, agonists or antagonists of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat: tetanus, diptheria, botulism, and/or meningitis type B.

[1030] Moreover, parasitic agents causing disease or symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardias, Helminthiasis, Leishmaniasis, Schistisoma, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose malaria.

[1031] Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.

[1032] Regeneration

[1033] Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59-87 (1997)). The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.

[1034] Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis.

[1035] Moreover, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.

[1036] Similarly, nerve and brain tissue could also be regenerated by using polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, to proliferate and differentiate nerve cells. Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated using the polynucleotides or polypeptides, as well as agonists or antagonists of the present invention.

[1037] Gastrointestinal Disorders

[1038] Polynucleotides or polypeptides, or agonists or antagonists of the present invention, may be used to treat, prevent, diagnose, and/or prognose gastrointestinal disorders, including inflammatory diseases and/or conditions, infections, cancers (e.g., intestinal neoplasms (carcinoid tumor of the small intestine, non-Hodgkin's lymphoma of the small intestine, small bowl lymphoma)), and ulcers, such as peptic ulcers.

[1039] Gastrointestinal disorders include dysphagia, odynophagia, inflammation of the esophagus, peptic esophagitis, gastric reflux, submucosal fibrosis and stricturing, Mallory-Weiss lesions, leiomyomas, lipomas, epidermal cancers, adeoncarcinomas, gastric retention disorders, gastroenteritis, gastric atrophy, gastric/stomach cancers, polyps of the stomach, autoimmune disorders such as pernicious anemia, pyloric stenosis, gastritis (bacterial, viral, eosinophilic, stress-induced, chronic erosive, atrophic, plasma cell, and Ménétrier's), and peritoneal diseases (e.g., chyloperioneum, hemoperitoneum, mesenteric cyst, mesenteric lymphadenitis, mesenteric vascular occlusion, panniculitis, neoplasms, peritonitis, pneumoperitoneum, bubphrenic abscess,).

[1040] Gastrointestinal disorders also include disorders associated with the small intestine, such as malabsorption syndromes, distension, irritable bowel syndrome, sugar intolerance, celiac disease, duodenal ulcers, duodenitis, tropical sprue, Whipple's disease, intestinal lymphangiectasia, Crohn's disease, appendicitis, obstructions of the ileum, Meckel's diverticulum, multiple diverticula, failure of complete rotation of the small and large intestine, lymphoma, and bacterial and parasitic diseases (such as Traveler's diarrhea, typhoid and paratyphoid, cholera, infection by Roundworms (Ascariasis lumbricoides), Hookworms (Ancylostoma duodenale), Threadworms (Enterobius vermicularis), Tapeworms (Taenia saginata, Echinococcus granulosus, Diphyllobothrium spp., and T. solium).

[1041] Liver diseases and/or disorders include intrahepatic cholestasis (alagille syndrome, biliary liver cirrhosis), fatty liver (alcoholic fatty liver, reye syndrome), hepatic vein thrombosis, hepatolentricular degeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension (esophageal and gastric varices), liver abscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary and experimental), alcoholic liver diseases (fatty liver, hepatitis, cirrhosis), parasitic (hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (hemolytic, hepatocellular, and cholestatic), cholestasis, portal hypertension, liver enlargement, ascites, hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune, hepatitis B, hepatitis C, hepatitis D, drug induced), toxic hepatitis, viral human hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), Wilson's disease, granulomatous hepatitis, secondary biliary cirrhosis, hepatic encephalopathy, portal hypertension, varices, hepatic encephalopathy, primary biliary cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma, hemangiomas, bile stones, liver failure (hepatic encephalopathy, acute liver failure), and liver neoplasms (angiomyolipoma, calcified liver metastases, cystic liver metastases, epithelial tumors, fibrolamellar hepatocarcinoma, focal nodular hyperplasia, hepatic adenoma, hepatobiliary cystadenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma, liver cancer, liver hemangioendothelioma, mesenchymal hamartoma, mesenchymal tumors of liver, nodular regenerative hyperplasia, benign liver tumors (Hepatic cysts [Simple cysts, Polycystic liver disease, Hepatobiliary cystadenoma, Choledochal cyst], Mesenchymal tumors [Mesenchymal hamartoma, Infantile hemangioendothelioma, Hemangioma, Peliosis hepatis, Lipomas, Inflammatory pseudotumor, Miscellaneous], Epithelial tumors [Bile duct epithelium (Bile duct hamartoma, Bile duct adenoma), Hepatocyte (Adenoma, Focal nodular hyperplasia, Nodular regenerative hyperplasia)], malignant liver tumors [hepatocellular, hepatoblastoma, hepatocellular carcinoma, cholangiocellular, cholangiocarcinoma, cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi's sarcoma, hemangioendothelioma, other tumors, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous carcinoma, primary lymphoma]), peliosis hepatis, erythrohepatic porphyria, hepatic porphyria (acute intermittent porphyria, porphyria cutanea tarda), Zellweger syndrome).

[1042] Pancreatic diseases and/or disorders include acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis), neoplasms (adenocarcinoma of the pancreas, cystadenocarcinoma, insulinoma, gastrinoma, and glucagonoma, cystic neoplasms, islet-cell tumors, pancreoblastoma), and other pancreatic diseases (e.g., cystic fibrosis, cyst (pancreatic pseudocyst, pancreatic fistula, insufficiency)).

[1043] Gallbladder diseases include gallstones (cholelithiasis and choledocholithiasis), postcholecystectomy syndrome, diverticulosis of the gallbladder, acute cholecystitis, chronic cholecystitis, bile duct tumors, and mucocele.

[1044] Diseases and/or disorders of the large intestine include antibiotic-associated colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscesses, fungal and bacterial infections, anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases (colitis, colonic neoplasms [colon cancer, adenomatous colon polyps (e.g., villous adenoma), colon carcinoma, colorectal cancer], colonic diverticulitis, colonic diverticulosis, megacolon [Hirschsprung disease, toxic megacolon]; sigmoid diseases [proctocolitis, sigmoin neoplasms]), constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery), duodenal diseases (duodenal neoplasms, duodenal obstruction, duodenal ulcer, duodenitis), enteritis (enterocolitis), HIV enteropathy, ileal diseases (ileal neoplasms, ileitis), immunoproliferative small intestinal disease, inflammatory bowel disease (ulcerative colitis, Crohn's disease), intestinal atresia, parasitic diseases (anisakiasis, balantidiasis, blastocystis infections, cryptosporidiosis, dientamoebiasis, amebic dysentery, giardiasis), intestinal fistula (rectal fistula), intestinal neoplasms (cecal neoplasms, colonic neoplasms, duodenal neoplasms, ileal neoplasms, intestinal polyps, jejunal neoplasms, rectal neoplasms), intestinal obstruction (afferent loop syndrome, duodenal obstruction, impacted feces, intestinal pseudo-obstruction [cecal volvulus], intussusception), intestinal perforation, intestinal polyps (colonic polyps, gardner syndrome, peutz-jeghers syndrome), jejunal diseases (jejunal neoplasms), malabsorption syndromes (blind loop syndrome, celiac disease, lactose intolerance, short bowl syndrome, tropical sprue, whipple's disease), mesenteric vascular occlusion, pneumatosis cystoides intestinalis, protein-losing enteropathies (intestinal lymphagiectasis), rectal diseases (anus diseases, fecal incontinence, hemorrhoids, proctitis, rectal fistula, rectal prolapse, rectocele), peptic ulcer (duodenal ulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer, Zollinger-Ellison syndrome), postgastrectomy syndromes (dumping syndrome), stomach diseases (e.g., achlorhydria, duodenogastric reflux (bile reflux), gastric antral vascular ectasia, gastric fistula, gastric outlet obstruction, gastritis (atrophic or hypertrophic), gastroparesis, stomach dilatation, stomach diverticulum, stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, hyperplastic gastric polyp), stomach rupture, stomach ulcer, stomach volvulus), tuberculosis, visceroptosis, vomiting (e.g., hematemesis, hyperemesis gravidarum, postoperative nausea and vomiting) and hemorrhagic colitis.

[1045] Further diseases and/or disorders of the gastrointestinal system include biliary tract diseases, such as, gastroschisis, fistula (e.g., biliary fistula, esophageal fistula, gastric fistula, intestinal fistula, pancreatic fistula), neoplasms (e.g., biliary tract neoplasms, esophageal neoplasms, such as adenocarcinoma of the esophagus, esophageal squamous cell carcinoma, gastrointestinal neoplasms, pancreatic neoplasms, such as adenocarcinoma of the pancreas, mucinous cystic neoplasm of the pancreas, pancreatic cystic neoplasms, pancreatoblastoma, and peritoneal neoplasms), esophageal disease (e.g., bullous diseases, candidiasis, glycogenic acanthosis, ulceration, barrett esophagus varices, atresia, cyst, diverticulum (e.g., Zenker's diverticulum), fistula (e.g., tracheoesophageal fistula), motility disorders (e.g., CREST syndrome, deglutition disorders, achalasia, spasm, gastroesophageal reflux), neoplasms, perforation (e.g., Boerhaave syndrome, Mallory-Weiss syndrome), stenosis, esophagitis, diaphragmatic hernia (e.g., hiatal hernia); gastrointestinal diseases, such as, gastroenteritis (e.g., cholera morbus, norwalk virus infection), hemorrhage (e.g., hematemesis, melena, peptic ulcer hemorrhage), stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, stomach cancer)), hernia (e.g., congenital diaphragmatic hernia, femoral hernia, inguinal hernia, obturator hernia, umbilical hernia, ventral hernia), and intestinal diseases (e.g., cecal diseases (appendicitis, cecal neoplasms)).

[1046] Chemotaxis

[1047] Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.

[1048] Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.

[1049] It is also contemplated that polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention could be used as an inhibitor of chemotaxis.

[1050] Binding Activity

[1051] A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.

[1052] Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991)). Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques.

[1053] Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.

[1054] The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.

[1055] Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.

[1056] Preferably, an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.

[1057] Additionally, the receptor to which the polypeptide of the present invention binds can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slides are exposed to the polypeptide of the present invention, after they have been labeled. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase.

[1058] Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.

[1059] As an alternative approach for receptor identification, the labeled polypeptides can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.

[1060] Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”) may be employed to modulate the activities of the polypeptide of the present invention thereby effectively generating agonists and antagonists of the polypeptide of the present invention. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998); each of these patents and publications are hereby incorporated by reference). In one embodiment, alteration of polynucleotides and corresponding polypeptides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired molecule by homologous, or site-specific, recombination. In another embodiment, polynucleotides and corresponding polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of the polypeptide of the present invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are family members. In further preferred embodiments, the heterologous molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).

[1061] Other preferred fragments are biologically active fragments of the polypeptide of the present invention. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.

[1062] Additionally, this invention provides a method of screening compounds to identify those which modulate the action of the polypeptide of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, a the polypeptide of the present invention, the compound to be screened and ³[H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate. A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates proliferation by determining the uptake of ³[H] thymidine in each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of ³[H] thymidine. Both agonist and antagonist compounds may be identified by this procedure.

[1063] In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound. The ability of the compound to enhance or block this interaction could then be measured. Alternatively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential agonist or antagonist. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

[1064] All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptides of the invention from suitably manipulated cells or tissues.

[1065] Therefore, the invention includes a method of identifying compounds which bind to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the present invention; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with a polypeptide of the present invention, (b) assaying a biological activity, and (b) determining if a biological activity of the polypeptide has been altered.

[1066] Targeted Delivery

[1067] In another embodiment, the invention provides a method of delivering compositions to targeted cells expressing a receptor for a polypeptide of the invention, or cells expressing a cell bound form of a polypeptide of the invention.

[1068] As discussed herein, polypeptides or antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.

[1069] In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., polypeptides of the invention or antibodies of the invention) in association with toxins or cytotoxic prodrugs.

[1070] By “toxin” is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

[1071] Drug Screening

[1072] Further contemplated is the use of the polypeptides of the present invention, or the polynucleotides encoding these polypeptides, to screen for molecules which modify the activities of the polypeptides of the present invention. Such a method would include contacting the polypeptide of the present invention with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of these polypeptides following binding.

[1073] This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and a polypeptide of the present invention.

[1074] Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the present invention. These methods comprise contacting such an agent with a polypeptide of the present invention or a fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or a fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the polypeptides of the present invention.

[1075] Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the present invention, and is described in great detail in European Patent Application 84/03564, published on Sep. 13, 1984, which is incorporated herein by reference herein. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with polypeptides of the present invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.

[1076] This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the present invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with a polypeptide of the invention.

[1077] Polypeptides of the Invention Binding Peptides and Other Molecules

[1078] The invention also encompasses screening methods for identifying polypeptides and nonpolypeptides that bind polypeptides of the invention, and the polypeptide of the invention binding molecules identified thereby. These binding molecules are useful, for example, as agonists and antagonists of the polypeptides of the invention. Such agonists and antagonists can be used, in accordance with the invention, in the therapeutic embodiments described in detail, below.

[1079] This method comprises the steps of:contacting a polypeptide of the invention with a plurality of molecules; and identifying a molecule that binds the polypeptide of the invention.

[1080] The step of contacting the polypeptide of the invention with the plurality of molecules may be effected in a number of ways. For example, one may contemplate immobilizing the polypeptide of the invention on a solid support and bringing a solution of the plurality of molecules in contact with the immobilized polypeptide of the invention. Such a procedure would be akin to an affinity chromatographic process, with the affinity matrix being comprised of the immobilized polypeptide of the invention. The molecules having a selective affinity for the polypeptide of the invention can then be purified by affinity selection. The nature of the solid support, process for attachment of the polypeptide of the invention to the solid support, solvent, and conditions of the affinity isolation or selection are largely conventional and well known to those of ordinary skill in the art.

[1081] Alternatively, one may also separate a plurality of polypeptides into substantially separate fractions comprising a subset of or individual polypeptides. For instance, one can separate the plurality of polypeptides by gel electrophoresis, column chromatography, or like method known to those of ordinary skill for the separation of polypeptides. The individual polypeptides can also be produced by a transformed host cell in such a way as to be expressed on or about its outer surface (e.g., a recombinant phage). Individual isolates can then be “probed” by the polypeptide of the invention, optionally in the presence of an inducer should one be required for expression, to determine if any selective affinity interaction takes place between the polypeptide of the invention and the individual clone. Prior to contacting the polypeptide of the invention with each fraction comprising individual polypeptides, the polypeptides could first be transferred to a solid support for additional convenience. Such a solid support may simply be a piece of filter membrane, such as one made of nitrocellulose or nylon. In this manner, positive clones could be identified from a collection of transformed host cells of an expression library, which harbor a DNA construct encoding a polypeptide having a selective affinity for a polypeptide of the invention. Furthermore, the amino acid sequence of the polypeptide having a selective affinity for the polypeptide of the invention can be determined directly by conventional means or the coding sequence of the DNA encoding the polypeptide can frequently be determined more conveniently. The primary sequence can then be deduced from the corresponding DNA sequence. If the amino acid sequence is to be determined from the polypeptide itself, one may use microsequencing techniques. The sequencing technique may include mass spectroscopy.

[1082] In certain situations, it may be desirable to wash away any unbound polypeptide of the invention, or alterntatively, unbound polypeptides, from a mixture of the polypeptide of the invention and the plurality of polypeptides prior to attempting to determine or to detect the presence of a selective affinity interaction. Such a wash step may be particularly desirable when the polypeptide of the invention or the plurality of polypeptides is bound to a solid support.

[1083] The plurality of molecules provided according to this method may be provided by way of diversity libraries, such as random or combinatorial peptide or nonpeptide libraries which can be screened for molecules that specifically bind to a polypeptide of the invention. Many libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries. Examples of chemically synthesized libraries are described in Fodor et al., 1991, Science 251:767-773; Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology 12:709-710;Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.

[1084] Examples of phage display libraries are described in Scott and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

[1085] In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991; and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.

[1086] By way of examples of nonpeptide libraries, a benzodiazepine library (see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712) can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).

[1087] The variety of non-peptide libraries that are useful in the present invention is great. For example, Ecker and Crooke, 1995, Bio/Technology 13:351-360 list benzodiazepines, hydantoins, piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, and oxazolones as among the chemical species that form the basis of various libraries.

[1088] Non-peptide libraries can be classified broadly into two types: decorated monomers and oligomers. Decorated monomer libraries employ a relatively simple scaffold structure upon which a variety functional groups is added. Often the scaffold will be a molecule with a known useful pharmacological activity. For example, the scaffold might be the benzodiazepine structure.

[1089] Non-peptide oligomer libraries utilize a large number of monomers that are assembled together in ways that create new shapes that depend on the order of the monomers. Among the monomer units that have been used are carbamates, pyrrolinones, and morpholinos. Peptoids, peptide-like oligomers in which the side chain is attached to the alpha amino group rather than the alpha carbon, form the basis of another version of non-peptide oligomer libraries. The first non-peptide oligomer libraries utilized a single type of monomer and thus contained a repeating backbone. Recent libraries have utilized more than one monomer, giving the libraries added flexibility.

[1090] Screening the libraries can be accomplished by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al.; Rebar and Pabo, 1993, Science 263:671-673; and CT Publication No. WO 94/18318.

[1091] In a specific embodiment, screening to identify a molecule that binds a polypeptide of the invention can be carried out by contacting the library members with a polypeptide of the invention immobilized on a solid phase and harvesting those library members that bind to the polypeptide of the invention. Examples of such screening methods, termed “panning” techniques are described by way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and in references cited herein.

[1092] In another embodiment, the two-hybrid system for selecting interacting proteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used to identify molecules that specifically bind to a polypeptide of the invention.

[1093] Where the polypeptide of the invention binding molecule is a polypeptide, the polypeptide can be conveniently selected from any peptide library, including random peptide libraries, combinatorial peptide libraries, or biased peptide libraries. The term “biased” is used herein to mean that the method of generating the library is manipulated so as to restrict one or more parameters that govern the diversity of the resulting collection of molecules, in this case peptides.

[1094] Thus, a truly random peptide library would generate a collection of peptides in which the probability of finding a particular amino acid at a given position of the peptide is the same for all 20 amino acids. A bias can be introduced into the library, however, by specifying, for example, that a lysine occur every fifth amino acid or that positions 4, 8, and 9 of a decapeptide library be fixed to include only arginine. Clearly, many types of biases can be contemplated, and the present invention is not restricted to any particular bias. Furthermore, the present invention contemplates specific types of peptide libraries, such as phage displayed peptide libraries and those that utilize a DNA construct comprising a lambda phage vector with a DNA insert.

[1095] As mentioned above, in the case of a polypeptide of the invention binding molecule that is a polypeptide, the polypeptide may have about 6 to less than about 60 amino acid residues, preferably about 6 to about 10 amino acid residues, and most preferably, about 6 to about 22 amino acids. In another embodiment, a polypeptide of the invention binding polypeptide has in the range of 15-100 amino acids, or 20-50 amino acids.

[1096] The selected polypeptide of the invention binding polypeptide can be obtained by chemical synthesis or recombinant expression.

[1097] Antisense and Ribozyme (Antagonists)

[1098] In specific embodiments, antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO: X, or the complementary strand thereof, and/or to nucleotide sequences contained a deposited clone. In one embodiment, antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as Anitsense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed for example, in Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research, 6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science, 251:1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.

[1099] For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines was previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments were performed in vitro by incubating cells with the oligoribonucleotide. A similar procedure for in vivo use is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is produced as follows: A sequence complimentary to the first 15 bases of the open reading frame is flanked by an EcoR1 site on the 5 end and a HindIII site on the 3 end. Next, the pair of oligonucleotides is heated at 90° C. for one minute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5, 10 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated to the EcoR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).

[1100] For example, the 5′ coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.

[1101] In one embodiment, the antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the antisense nucleic acid of the invention. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding a polypeptide of the invention, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster et al., Nature, 296:39-42 (1982)), etc.

[1102] The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of interest. However, absolute complementarity, although preferred, is not required. A sequence “complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids of the invention, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence of the invention it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

[1103] Oligonucleotides that are complementary to the 5′ end of the message, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3′ untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., Nature, 372:333-335 (1994). Thus, oligonucleotides complementary to either the 5′- or 3′-non-translated, non-coding regions of a polynucleotide sequence of the invention could be used in an antisense approach to inhibit translation of endogenous mRNA. Oligonucleotides complementary to the 5′ untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5′-, 3′- or coding region of mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

[1104] The polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-652 (1987); PCT Publication NO: WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication NO: WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al., BioTechniques, 6:958-976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res., 5:539-549 (1988)). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

[1105] The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

[1106] The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[1107] In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

[1108] In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al., Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a 2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215:327-330 (1987)).

[1109] Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (Nucl. Acids Res., 16:3209 (1988)), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A., 85:7448-7451 (1988)), etc.

[1110] While antisense nucleotides complementary to the coding region sequence of the invention could be used, those complementary to the transcribed untranslated region are most preferred.

[1111] Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published Oct. 4, 1990; Sarver et al, Science, 247:1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy mRNAs corresponding to the polynucleotides of the invention, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5′-UG-3′. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage sites within each nucleotide sequence disclosed in the sequence listing. Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5′ end of the mRNA corresponding to the polynucleotides of the invention; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

[1112] As in the antisense approach, the ribozymes of the invention can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express the polynucleotides of the invention in vivo. DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. A preferred method of delivery involves using a DNA construct “encoding” the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

[1113] Antagonist/agonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the present invention on neoplastic cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth.

[1114] The antagonist/agonist may also be employed to prevent hyper-vascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty.

[1115] The antagonist/agonist may also be employed to prevent the growth of scar tissue during wound healing.

[1116] The antagonist/agonist may also be employed to treat, prevent, and/or diagnose the diseases described herein.

[1117] Thus, the invention provides a method of treating or preventing diseases, disorders, and/or conditions, including but not limited to the diseases, disorders, and/or conditions listed throughout this application, associated with overexpression of a polynucleotide of the present invention by administering to a patient (a) an antisense molecule directed to the polynucleotide of the present invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention.

[1118] invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention

[1119] Other Activities

[1120] The polypeptide of the present invention, as a result of the ability to stimulate vascular endothelial cell growth, may be employed in treatment for stimulating re-vascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. These polypeptide may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.

[1121] The polypeptide may also be employed for treating wounds due to injuries, burns, post-operative tissue repair, and ulcers since they are mitogenic to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.

[1122] The polypeptide of the present invention may also be employed stimulate neuronal growth and to treat, prevent, and/or diagnose neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. The polypeptide of the invention may have the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid in tissue transplants or bone grafts.

[1123] The polypeptide of the present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.

[1124] The polypeptide of the invention may also be employed for preventing hair loss, since FGF family members activate hair-forming cells and promotes melanocyte growth. Along the same lines, the polypeptides of the present invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.

[1125] The polypeptide of the invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues.

[1126] The polypeptide of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.

[1127] The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.

[1128] The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, polypeptides or polynucleotides and/or agonist or antagonists of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.

[1129] A polypeptide, polynucleotide, agonist, or antagonist of the present invention may be used to treat weight disorders, including but not limited to, obesity, cachexia, wasting disease, anorexia, and bulimia.

[1130] Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, caricadic rhythms, depression (including depressive diseases, disorders, and/or conditions), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.

[1131] Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.

[1132] Other Preferred Embodiments

[1133] Other preferred embodiments of the claimed invention include an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: X wherein X is any integer as defined in Table 1A. Also preferred is the above nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO: X in the range of positions beginning with the nucleotide at about the position of the 5′ Nucleotide of the Clone Sequence and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO: X in Table 1A. Further preferred is the above nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO: X in the range of positions beginning with the nucleotide at about the position of the 5′ Nucleotide of the Start Codon and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO: X in Table 1A. Similarly preferred is the above nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO: X in the range of positions beginning with the nucleotide at about the position of the 5′ Nucleotide of the First Amino Acid of the Signal Peptide and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO: X in Table 1A.

[1134] Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: X.

[1135] Further preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: X.

[1136] A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NO: X beginning with the nucleotide at about the position of the 5′ Nucleotide of the First Amino Acid of the Signal Peptide and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO: X in Table 1A.

[1137] A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ID NO: X.

[1138] Also preferred is an isolated nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule, wherein said isolated nucleic acid molecule does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.

[1139] Also preferred is a composition of matter comprising a DNA molecule which comprises a human cDNA clone identified by a cDNA Clone Identifier in Table 1A, which DNA molecule is contained in the material deposited with the American Type Culture Collection and given the ATCC Deposit Number shown in Table 1A for said cDNA Clone Identifier.

[1140] Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the nucleotide sequence of the cDNA of a human cDNA clone identified by a cDNA Clone Identifier in Table 1A, which DNA molecule is contained in the deposit given the ATCC Deposit Number shown in Table 1A. Further preferred is the above nucleic acid molecule, wherein said sequence of at least 50 contiguous nucleotides is included in the nucleotide sequence of the complete open reading frame sequence encoded by said human cDNA clone. In addition, an isolated nucleic acid molecule of the invention may comprise a nucleotide sequence which is at least 95% identical to sequence of at least 150 contiguous nucleotides in the nucleotide sequence of the cDNA in said human cDNA clone. A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 500 contiguous nucleotides in the nucleotide sequence of the cDNA in said human cDNA clone. A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of the cDNA in said human cDNA clone.

[1141] A further preferred embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO: X wherein X is any integer as defined in Table 1A; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A; which method comprises: (a) a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample with a sequence selected from said group; and (b) determining whether the sequence of said nucleic acid molecule in said sample is at least 95% identical to said selected sequence. The step of comparing sequences in the above method may further comprise determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group. Similarly, the step of comparing sequences in the above method may be performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

[1142] A further preferred embodiment is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO: X wherein X is any integer as defined in Table 1A; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A. This method described above may further comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.

[1143] Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene encoding a secreted protein identified in Table 1A, which method comprises a step of detecting in a biological sample obtained from said subject nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO: X wherein X is any integer as defined in Table 1A; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A. This method described above may further comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.

[1144] Also preferred is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO: X wherein X is any integer as defined in Table 1A; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

[1145] Also preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the amino acid sequence of SEQ ID NO: Y wherein Y is any integer as defined in Table 1A. Further preferred is the above isolated polypeptide, wherein said sequence of contiguous amino acids is included in the amino acid sequence of SEQ ID NO: Y in the range of positions beginning with the residue at about the position of the First Amino Acid of the Secreted Portion and ending with the residue at about the Last Amino Acid of the Open Reading Frame as set forth for SEQ ID NO: Y in Table 1A.

[1146] Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of SEQ ID NO: Y.

[1147] Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of SEQ ID NO: Y.

[1148] Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the complete amino acid sequence of SEQ ID NO: Y.

[1149] Also preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A. Further preferred is the above isolated polypeptide wherein said sequence of contiguous a-amino acids is included in the amino acid sequence of a secreted portion of the secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A.

[1150] Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A.

[1151] Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A.

[1152] Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A.

[1153] Further preferred is an isolated antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO: Y wherein Y is any integer as defined in Table 1A; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A.

[1154] Further preferred is a method for detecting in a biological sample a polypeptide comprising an amino acid sequence which is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO: Y wherein Y is any integer as defined in Table 1A; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A; which method comprises: (a) a step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group; and (b) determining whether the sequence of said polypeptide molecule in said sample is at least 90% identical to said sequence of at least 10 contiguous amino acids. The step in the above method of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group may further comprise determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO: Y wherein Y is any integer as defined in Table 1A; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A. Further, the step of comparing sequences in the above method may be performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.

[1155] Also preferred is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO: Y wherein Y is any integer as defined in Table 1A; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A. This method may further comprise a step of detecting polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the above group.

[1156] Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene encoding a secreted protein identified in Table 1A, which method comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO: Y wherein Y is any integer as defined in Table 1A; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A.

[1157] In any of these methods, the step of detecting said polypeptide molecules includes using an antibody.

[1158] Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO: Y wherein Y is any integer as defined in Table 1A; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A. Further preferred is the above isolated nucleic acid molecule, wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host. Similarly preferred is the above isolated nucleic acid molecule, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO: Y wherein Y is any integer as defined in Table 1A; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A.

[1159] Further preferred is a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecules into a vector. Also preferred is the recombinant vector produced by this method. Also preferred is a method of making a recombinant host cell comprising introducing the vector of the invention into a host cell, as well as the recombinant host cell produced by this method.

[1160] Also preferred is a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a secreted portion of a human secreted protein comprising an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO: Y beginning with the residue at the position of the First Amino Acid of the Secreted Portion of SEQ ID NO: Y wherein Y is an integer set forth in Table 1A and said position of the First Amino Acid of the Secreted Portion of SEQ ID NO: Y is defined in Table 1A; and an amino acid sequence of a secreted portion of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1A. The isolated polypeptide produced by this method is also preferred.

[1161] Also preferred is a method of treatment of an individual in need of an increased level of a secreted protein activity, which method comprises administering to such an individual a pharmaceutical composition comprising an amount of an isolated polypeptide, polynucleotide, or antibody of the claimed invention effective to increase the level of said protein activity in said individual.

[1162] Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

[1163] The above-recited applications have uses in a wide variety of hosts. Such hosts include, but are not limited to, human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, and human. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In most preferred embodiments, the host is a human.

[1164] Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

EXAMPLES Example 1

[1165] Isolation of a Selected cDNA Clone From the Deposited Sample

[1166] Each cDNA clone in a cited ATCC deposit is contained in a plasmid vector. Table 1A identifies the vectors used to construct the cDNA library from which each clone was isolated. In many cases, the vector used to construct the library is a phage vector from which a plasmid has been excised. The table immediately below correlates the related plasmid for each phage vector used in constructing the cDNA library. For example, where a particular clone is identified in Table 1A as being isolated in the vector “Lambda Zap,” the corresponding deposited clone is in “pBluescript.” Vector Used to Construct Library Corresponding Deposited Plasmid Lambda Zap pBluescript (pBS) Uni-Zap XR pBluescript (pBS) Zap Express pBK lafmid BA plafmid BA pSport1 pSport1 pCMVSport 2.0 pCMVSport 2.0 pCMVSport 3.0 pCMVSport 3.0 pCR ® 2.1 pCR ® 2.1

[1167] Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Both can be transformed into E. coli strain XL-1 Blue, also available from Stratagene. pBS comes in 4 forms SK+, SK−, KS+ and KS. The S and K refers to the orientation of the polylinker to the T7 and T3 primer sequences which flank the polylinker region (“S ” is for SacI and “K” is for KpnI which are the first sites on each respective end of the linker). “+” or “−” refer to the orientation of the f1 origin of replication (“ori”), such that in one orientation, single stranded rescue initiated from the f1 ori generates sense strand DNA and in the other, antisense.

[1168] Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, Inc., P. O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from Life Technologies. (See, for instance, Gruber, C. E., et al., Focus 15:59 (1993).) Vector lafmid BA (Bento Soares, Columbia University, N.Y.) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. (See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).) Preferably, a polynucleotide of the present invention does not comprise the phage vector sequences identified for the particular clone in Table 1A, as well as the corresponding plasmid vector sequences designated above.

[1169] The deposited material in the sample assigned the ATCC Deposit Number cited in Table 1A for any given cDNA clone also may contain one or more additional plasmids, each comprising a cDNA clone different from that given clone. Thus, deposits sharing the same ATCC Deposit Number contain at least a plasmid for each cDNA clone identified in Table 1A. Typically, each ATCC deposit sample cited in Table 1A comprises a mixture of approximately equal amounts (by weight) of about 50 plasmid DNAs, each containing a different cDNA clone; but such a deposit sample may include plasmids for more or less than 50 cDNA clones, up to about 500 cDNA clones.

[1170] Two approaches can be used to isolate a particular clone from the deposited sample of plasmid DNAs cited for that clone in Table 1A. First, a plasmid is directly isolated by screening the clones using a polynucleotide probe corresponding to SEQ ID NO: X.

[1171] Particularly, a specific polynucleotide with 30-40 nucleotides is synthesized using an Applied Biosystems DNA synthesizer according to the sequence reported. The oligonucleotide is labeled, for instance, with ³²P-γ-ATP using T4 polynucleotide kinase and purified according to routine methods. (E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmid mixture is transformed into a suitable host, as indicated above (such as XL-1 Blue (Stratagene)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents cited above. The transformants are plated on 1.5% agar plates (containing the appropriate selection agent, e.g., ampicillin) to a density of about 150 transformants (colonies) per plate. These plates are screened using Nylon membranes according to routine methods for bacterial colony screening (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to 1.104), or other techniques known to those of skill in the art.

[1172] Alternatively, two primers of 17-20 nucleotides derived from both ends of the SEQ ID NO: X (i.e., within the region of SEQ ID NO: X bounded by the 5′ NT and the 3′ NT of the clone defined in Table 1A) are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 ul of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5-5 MM MgCl₂, 0.01% (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at 55 degree C. for 1 min; elongation at 72 degree C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.

[1173] Several methods are available for the identification of the 5′ or 3′ non-coding portions of a gene which may not be present in the deposited clone. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5′ and 3′ “RACE” protocols which are well known in the art. For instance, a method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length transcript. (Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684 (1993).)

[1174] Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably containing full-length gene RNA transcripts. A primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5′ portion of the desired full-length gene. This amplified product may then be sequenced and used to generate the full length gene.

[1175] This above method starts with total RNA isolated from the desired source, although poly-A+ RNA can be used. The RNA preparation can then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.

[1176] This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the desired gene.

Example 2

[1177] Isolation of Genomic Clones Corresponding to a Polynucleotide

[1178] A human genomic P1 library (Genomic Systems, Inc.) is screened by PCR using primers selected for the cDNA sequence corresponding to SEQ ID NO: X., according to the method described in Example 1. (See also, Sambrook.)

Example 3

[1179] Tissue Distribution of Polypeptide

[1180] Tissue distribution of mRNA expression of polynucleotides of the present invention is determined using protocols for Northern blot analysis, described by, among others, Sambrook et al. For example, a cDNA probe produced by the method described in Example 1 is labeled with P³² using the rediprime™ DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using CHROMA SPIN-100™ column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The purified labeled probe is then used to examine various human tissues for mRNA expression.

[1181] Multiple Tissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) (Clontech) are examined with the labeled probe using ExpressHyb™ hybridization solution (Clontech) according to manufacturer's protocol number PT1190-1. Following hybridization and washing, the blots are mounted and exposed to film at −70 degree C. overnight, and the films developed according to standard procedures.

Example 4

[1182] Chromosomal Mapping of the Polynucleotides

[1183] An oligonucleotide primer set is designed according to the sequence at the 5′ end of SEQ ID NO: X. This primer preferably spans about 100 nucleotides. This primer set is then used in a polymerase chain reaction under the following set of conditions: 30 seconds, 95 degree C.; 1 minute, 56 degree C.; 1 minute, 70 degree C. This cycle is repeated 32 times followed by one 5 minute cycle at 70 degree C. Human, mouse, and hamster DNA is used as template in addition to a somatic cell hybrid panel containing individual chromosomes or chromosome fragments (Bios, Inc). The reactions is analyzed on either 8% polyacrylamide gels or 3.5% agarose gels. Chromosome mapping is determined by the presence of an approximately 100 bp PCR fragment in the particular somatic cell hybrid.

Example 5

[1184] Bacterial Expression of a Polypeptide

[1185] A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 1, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5′ end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and XbaI correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic resistance (Amp^(r)), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.

[1186] The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kan^(r)). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.

[1187] Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression.

[1188] Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 6000×g). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at 4 degree C. The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid (“Ni—NTA”) affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6× His tag bind to the Ni—NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).

[1189] Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

[1190] The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni—NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM immidazole. Immidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4 degree C. or frozen at −80 degree C.

[1191] In addition to the above expression vector, the present invention further includes an expression vector comprising phage operator and promoter elements operatively linked to a polynucleotide of the present invention, called pHE4a. (ATCC Accession Number 209645, deposited on Feb. 25, 1998.) This vector contains: 1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence, and 6) the lactose operon repressor gene (lacIq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). The promoter sequence and operator sequences are made synthetically.

[1192] DNA can be inserted into the pHEa by restricting the vector with NdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted product on a gel, and isolating the larger fragment (the stuffer fragment should be about 310 base pairs). The DNA insert is generated according to the PCR protocol described in Example 1, using PCR primers having restriction sites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.

[1193] The engineered vector could easily be substituted in the above protocol to express protein in a bacterial system.

Example 6

[1194] Purification of a Polypeptide from an Inclusion Body

[1195] The following alternative method can be used to purify a polypeptide expressed in E coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4-10 degree C.

[1196] Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10 degree C. and the cells harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer.

[1197] The cells are then lysed by passing the solution through a microfluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000×g for 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.

[1198] The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×g centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4 degree C. overnight to allow further GuHCl extraction.

[1199] Following high speed centrifugation (30,000×g) to remove insoluble particles, the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded diluted protein solution is kept at 4 degree C. without mixing for 12 hours prior to further purification steps.

[1200] To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0.16 um membrane filter with appropriate surface area (e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.

[1201] Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion (Poros CM-20, Perseptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10 column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A₂₈₀ monitoring of the effluent. Fractions containing the polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.

[1202] The resultant polypeptide should exhibit greater than 95% purity after the above refolding and purification steps. No major contaminant bands should be observed from Commassie blue stained 16% SDS-PAGE gel when 5 ug of purified protein is loaded. The purified protein can also be tested for endotoxin/LPS contamination, and typically the LPS content is less than 0.1 ng/ml according to LAL assays.

Example 7

[1203] Cloning and Expression of a Polypeptide in a Baculovirus Expression System

[1204] In this example, the plasmid shuttle vector pA2 is used to insert a polynucleotide into a baculovirus to express a polypeptide. This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites such as BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40 (“SV40”) is used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned polynucleotide.

[1205] Many other baculovirus vectors can be used in place of the vector above, such as pAc373, pVL941, and pAcIM1, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al., Virology 170:31-39 (1989).

[1206] Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon and the naturally associated leader sequence identified in Table 1A, is amplified using the PCR protocol described in Example 1. If the naturally occurring signal sequence is used to produce the secreted protein, the pA2 vector does not need a second signal peptide. Alternatively, the vector can be modified (pA2 GP) to include a baculovirus leader sequence, using the standard methods described in Summers et al., “A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,” Texas Agricultural Experimental Station Bulletin No. 1555 (1987).

[1207] The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.

[1208] The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).

[1209] The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.

[1210] Five ug of a plasmid containing the polynucleotide is co-transfected with 1.0 ug of a commercially available linearized baculovirus DNA (“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of BaculoGold™ virus DNA and 5 ug of the plasmid are mixed in a sterile well of a microtiter plate containing 50 ul of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27 degrees C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27 degrees C. for four days.

[1211] After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10.) After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf) The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ul of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4 degree C.

[1212] To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection (“MOI”) of about 2. If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of ³⁵S-methionine and 5 uCi ³⁵S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

[1213] Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the produced protein.

Example 8

[1214] Expression of a Polypeptide in Mammalian Cells

[1215] The polypeptide of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMN). However, cellular elements can also be used (e.g., the human actin promoter).

[1216] Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

[1217] Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.

[1218] The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.

[1219] Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession No. 209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41:521-530 (1985).) Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of the gene of interest. The vectors also contain the 3′ intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under control of the SV40 early promoter.

[1220] Specifically, the plasmid pC6, for example, is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel.

[1221] A polynucleotide of the present invention is amplified according to the protocol outlined in Example 1. If the naturally occurring signal sequence is used to produce the secreted protein, the vector does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/348911.) The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean, ” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.

[1222] The amplified fragment is then digested with the same restriction enzyme and purified on a 1 % agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.

[1223] Chinese hamster ovary cells lacking an active DHFR gene is used for transfection. Five μ g of the expression plasmid pC6 a pC4 is cotransfected with 0.5 ug of the plasmid pSVneo using lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 uM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.

Example 9

[1224] Protein Fusions

[1225] The polypeptides of the present invention are preferably fused to other proteins. These fusion proteins can be used for a variety of applications. For example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See Example 5; see also EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusion to IgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclear localization signals fused to the polypeptides of the present invention can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function. Finally, fusion proteins can increase solubility and/or stability of the fused protein compared to the non-fused protein. All of the types of fusion proteins described above can be made by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule, or the protocol described in Example 5.

[1226] Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5′ and 3′ ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector.

[1227] For example, if pC4 (Accession No. 209646) is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3′ BamHI site should be destroyed. Next, the vector containing the human Fc portion is re-restricted with BamHI, linearizing the vector, and a polynucleotide of the present invention, isolated by the PCR protocol described in Example 1, is ligated into this BamHI site. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced.

[1228] If the naturally occurring signal sequence is used to produce the secreted protein, pC4 does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.) Human IgG Fe region: (SEQ ID NO:1) GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAA ACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGG TGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTG GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG TAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 10

[1229] Production of an Antibody from a Polypeptide

[1230] The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, cells expressing a polypeptide of the present invention is administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of the secreted protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.

[1231] In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or protein binding fragments thereof). Such monoclonal antibodies can be prepared using hybridoma technology. (Köhler et al., Nature 256:495 (1975); Köhler et al., Eur. J. Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In general, such procedures involve immunizing an animal (preferably a mouse) with polypeptide or, more preferably, with a secreted polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56 degrees C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.

[1232] The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP20), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the polypeptide.

[1233] Alternatively, additional antibodies capable of binding to the polypeptide can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the protein-specific antibody can be blocked by the polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein-specific antibody and can be used to immunize an animal to induce formation of further protein-specific antibodies.

[1234] It will be appreciated that Fab and F(ab′)2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). Alternatively, secreted protein-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.

[1235] For in vivo use of antibodies in humans, it may be preferable to use “humanized” chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).)

Example 11

[1236] Production Of Secreted Protein For High-Throughput Screening Assays

[1237] The following protocol produces a supernatant containing a polypeptide to be tested. This supernatant can then be used in the Screening Assays described herein.

[1238] First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution (1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker) for a working solution of 50 ug/ml. Add 200 ul of this solution to each well (24 well plates) and incubate at RT for 20 minutes. Be sure to distribute the solution over each well (note: a 12-channel pipetter may be used with tips on every other channel). Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS (Phosphate Buffered Saline). The PBS should remain in the well until just prior to plating the cells and plates may be poly-lysine coated in advance for up to two weeks.

[1239] Plate 293T cells (do not carry cells past P+20) at 2×10⁵ cells/well in 0.5ml DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G IL glucose and L-glutamine (12-604F Biowhittaker))/10% heat inactivated FBS(14-503F Biowhittaker)/1× Penstrep(17-602E Biowhittaker). Let the cells grow overnight.

[1240] The next day, mix together in a sterile solution basin: 300 ul Lipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070 Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter, aliquot approximately 2 ug of an expression vector containing a polynucleotide insert, produced by the methods described in Examples 8 or 9, into an appropriately labeled 96-well round bottom plate. With a multi-channel pipetter, add 50 ul of the Lipofectamine/Optimem I mixture to each well. Pipette up and down gently to mix. Incubate at RT 15-45 minutes. After about 20 minutes, use a multi-channel pipetter to add 150 ul Optimem I to each well. As a control, one plate of vector DNA lacking an insert should be transfected with each set of transfections.

[1241] Preferably, the transfection should be performed by tag-teaming the following tasks. By tag-teaming, hands on time is cut in half, and the cells do not spend too much time on PBS. First, person A aspirates off the media from four 24-well plates of cells, and then person B rinses each well with 0.5-1 ml PBS. Person A then aspirates off PBS rinse, and person B, using a 12-channel pipetter with tips on every other channel, adds the 200 ul of DNA/Lipofectamine/Optimem I complex to the odd wells first, then to the even wells, to each row on the 24-well plates. Incubate at 37 degrees C. for 6 hours.

[1242] While cells are incubating, prepare appropriate media, either 1% BSA in DMEM with 1× penstrep, or CHO-5 media (116.6 mg/L of CaCl2 (anhyd); 0.00130 mg/L CuSO₄-5H₂O; 0.050 mg/L of Fe(NO₃)₃-9H₂O; 0.417 mg/L of FeSO₄-7H₂O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl₂; 48.84 mg/L of MgSO₄; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO₃; 62.50 mg/L of NaH₂PO₄-H₂0; 71.02 mg/L of Na₂HPO₄; 0.4320 mg/L of ZnSO₄-7H₂O; 0.002 mg/L of Arachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L of DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L of Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551 mg/L of D-Glucose; 130.85 mg/ml of L-Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H₂0; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of L-Cystine-2HCL-H₂0; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml of L-Histidine-HCL-H₂0; 106.97 mg/ml of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine; 19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H₂0; 99.65 mg/ml of L-Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin; 3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; and 0.680 mg/L of Vitamin B1₂; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM of Ethanolamine; 0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrin complexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrin complexed with Oleic Acid; and 10 mg/L of Methyl-B-Cyclodextrin complexed with Retinal) with 2 mm glutamine and 1× penstrep. (BSA (81-068-3 Bayer) 100 gm dissolved in 1L DMEM for a 10% BSA stock solution). Filter the media and collect 50 ul for endotoxin assay in 15 ml polystyrene conical.

[1243] The transfection reaction is terminated, preferably by tag-teaming, at the end of the incubation period. Person A aspirates off the transfection media, while person B adds 1.5ml appropriate media to each well. Incubate at 37 degrees C. for 45 or 72 hours depending on the media used: 1% BSA for 45 hours or CHO-5 for 72 hours.

[1244] On day four, using a 300 ul multichannel pipetter, aliquot 600 ul in one 1 ml deep well plate and the remaining supernatant into a 2 ml deep well. The supernatants from each well can then be used in the assays described in Examples 13-20.

[1245] It is specifically understood that when activity is obtained in any of the assays described below using a supernatant, the activity originates from either the polypeptide directly (e.g., as a secreted protein) or by the polypeptide inducing expression of other proteins, which are then secreted into the supernatant. Thus, the invention further provides a method of identifying the protein in the supernatant characterized by an activity in a particular assay.

Example 12

[1246] Construction of GAS Reporter Construct

[1247] One signal transduction pathway involved in the differentiation and proliferation of cells is called the Jaks-STATs pathway. Activated proteins in the Jaks-STATs pathway bind to gamma activation site “GAS” elements or interferon-sensitive responsive element (“ISRE”), located in the promoter of many genes. The binding of a protein to these elements alter the expression of the associated gene.

[1248] GAS and ISRE elements are recognized by a class of transcription factors called Signal Transducers and Activators of Transcription, or “STATs.” There are six members of the STATs family. Stat1 and Stat3 are present in many cell types, as is Stat2 (as response to IFN-alpha is widespread). Stat4 is more restricted and is not in many cell types though it has been found in T helper class I, cells after treatment with IL-12. Stat5 was originally called mammary growth factor, but has been found at higher concentrations in other cells including myeloid cells. It can be activated in tissue culture cells by many cytokines.

[1249] The STATs are activated to translocate from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase (“Jaks”) family. Jaks represent a distinct family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinases display significant sequence similarity and are generally catalytically inactive in resting cells.

[1250] The Jaks are activated by a wide range of receptors summarized in the Table below. (Adapted from review by Schidler and Darnell, Ann. Rev. Biochem. 64:621-51 (1995).) A cytokine receptor family, capable of activating Jaks, is divided into two groups: (a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b) Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share a conserved cysteine motif (a set of four conserved cysteines and one tryptophan) and a WSXWS motif (a membrane proximal region encoding Trp-Ser-Xxx-Trp-Ser (SEQ ID NO: 2)).

[1251] Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn activate STATs, which then translocate and bind to GAS elements. This entire process is encompassed in the Jaks-STATs signal transduction pathway.

[1252] Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS or the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells. For example, growth factors and cytokines are known to activate the Jaks-STATs pathway. (See Table below.) Thus, by using GAS elements linked to reporter molecules, activators of the Jaks-STATs pathway can be identified. JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS (elements) or ISRE IFN family IFN-a/B + + − − 1, 2, 3 ISRE IFN-g + + − 1 GAS (IRF1 > Lys6 > IFP) Il-10 + ? ? − 1, 3 gp130 family IL-6 (Pleiotropic) + + + ? 1, 3 GAS (IRF1 > Lys6 > IFP) Il-11 (Pleiotropic) ? + ? ? 1, 3 OnM (Pleiotropic) ? + + ? 1, 3 LIF (Pleiotropic) ? + + ? 1, 3 CNTF (Pleiotropic) −/+ + + ? 1, 3 G-CSF (Pleiotropic) ? + ? ? 1, 3 IL-12 (Pleiotropic) + − + + 1, 3 g-C family IL-2 (lymphocytes) − + − + 1, 3, 5 GAS IL-4 (lymph/myeloid) − + − + 6 GAS (IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) − + − + 5 GAS IL-9 (lymphocytes) − + − + 5 GAS IL-13 (lymphocyte) − + ? ? 6 GAS IL-15 ? + ? + 5 GAS gp140 family IL-3 (myeloid) − − + − 5 GAS (IRF1 > IFP >> Ly6) IL-5 (myeloid) − − + − 5 GAS GM-CSF (myeloid) − − + − 5 GAS Growth hormone family GH ? − + − 5 PRL ? +/− + − 1, 3, 5 EPO ? − + − 5 GAS (B-CAS > LRF1 = IFP >> Ly6) Receptor Tyrosine Kinases EGF ? + + − 1, 3 GAS (IRF1) PDGF ? + + − 1, 3 CSF-1 ? + + − 1, 3 GAS (not IRF1)

[1253] To construct a synthetic GAS containing promoter element, which is used in the Biological Assays described in Examples 13-14, a PCR based strategy is employed to generate a GAS-SV40 promoter sequence. The 5′ primer contains four tandem copies of the GAS binding site found in the IRF1 promoter and previously demonstrated to bind STATs upon induction with a range of cytokines (Rothman et al., Immunity 1:457-468 (1994).), although other GAS or ISRE elements can be used instead. The 5′ primer also contains 18 bp of sequence complementary to the SV40 early promoter sequence and is flanked with an XhoI site. The sequence of the 5′ primer is: (SEQ ID NO:3) 5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCC CCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3′

[1254] The downstream primer is complementary to the SV40 promoter and is flanked with a Hind III site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO: 4)

[1255] PCR amplification is performed using the SV40 promoter template present in the B-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI/Hind III and subcloned into BLSK2-. (Stratagene.) Sequencing with forward and reverse primers confirms that the insert contains the following sequence: (SEQ ID NO:5) 5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGA AATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTC CCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCA TTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGG CCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGA GGCCTAGGCTTTTGCAAAAAGCTT:3′

[1256] With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2 reporter construct is next engineered. Here, the reporter molecule is a secreted alkaline phosphatase, or “SEAP.” Clearly, however, any reporter molecule can be instead of SEAP, in this or in any of the other Examples. Well known reporter molecules that can be used instead of SEAP include chloramphenicol acetyltransferase (CAT), luciferase, alkaline phosphatase, B-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody.

[1257] The above sequence confirmed synthetic GAS-SV40 promoter element is subcloned into the pSEAP-Promoter vector obtained from Clontech using HindIII and XhoI, effectively replacing the SV40 promoter with the amplified GAS:SV40 promoter element, to create the GAS-SEAP vector. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.

[1258] Thus, in order to generate mammalian stable cell lines expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using SalI and NotI, and inserted into a backbone vector containing the neomycin resistance gene, such as pGFP-1 (Clontech), using these restriction sites in the multiple cloning site, to create the GAS-SEAP/Neo vector. Once this vector is transfected into mammalian cells, this vector can then be used as a reporter molecule for GAS binding as described in Examples 13-14.

[1259] Other constructs can be made using the above description and replacing GAS with a different promoter sequence. For example, construction of reporter molecules containing NFK-B and EGR promoter sequences are described in Examples 15 and 16. However, many other promoters can be substituted using the protocols described in these Examples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, II-2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used to test reporter construct activity, such as HELA (epithelial), HUVEC (endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.

Example 13

[1260] High-Throughput Screening Assay for T-cell Activity.

[1261] The following protocol is used to assess T-cell activity by identifying factors, and determining whether supernate containing a polypeptide of the invention proliferates and/or differentiates T-cells. T-cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 12. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The T-cell used in this assay is Jurkat T-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4 cells (ATCC Accession No. CRL-1582) cells can also be used.

[1262] Jurkat T-cells are lymphoblastic CD4+Th1 helper cells. In order to generate stable cell lines, approximately 2 million Jurkat cells are transfected with the GAS-SEAP/neo vector using DMRIE-C (Life Technologies)(transfection procedure described below). The transfected cells are seeded to a density of approximately 20,000 cells per well and transfectants resistant to 1 mg/ml genticin selected. Resistant colonies are expanded and then tested for their response to increasing concentrations of interferon gamma. The dose response of a selected clone is demonstrated.

[1263] Specifically, the following protocol will yield sufficient cells for 75 wells containing 200 ul of cells. Thus, it is either scaled up, or performed in multiple to generate sufficient cells for multiple 96 well plates. Jurkat cells are maintained in RPMI+10% serum with 1% Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C and incubate at room temperature for 15-45 mins.

[1264] During the incubation period, count cell concentration, spin down the required number of cells (10⁷ per transfection), and resuspend in OPTI-MEM to a final concentration of 10⁷ cells/ml. Then add 1 ml of 1×10⁷ cells in OPTI-MEM to T25 flask and incubate at 37 degrees C. for 6 hrs. After the incubation, add 10 ml of RPMI+15% serum.

[1265] The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated with supernatants containing polypeptides of the invention and/or induced polypeptides of the invention as produced by the protocol described in Example 11.

[1266] On the day of treatment with the supernatant, the cells should be washed and resuspended in fresh RPMI+10% serum to a density of 500,000 cells per ml. The exact number of cells required will depend on the number of supernatants being screened. For one 96 well plate, approximately 10 million cells (for 10 plates, 100 million cells) are required.

[1267] Transfer the cells to a triangular reservoir boat, in order to dispense the cells into a 96 well dish, using a 12 channel pipette. Using a 12 channel pipette, transfer 200 ul of cells into each well (therefore adding 100,000 cells per well).

[1268] After all the plates have been seeded, 50 ul of the supernatants are transferred directly from the 96 well plate containing the supernatants into each well using a 12 channel pipette. In addition, a dose of exogenous interferon gamma (0.1, 1.0, 10 ng) is added to wells H9, H10, and H11 to serve as additional positive controls for the assay.

[1269] The 96 well dishes containing Jurkat cells treated with supernatants are placed in an incubator for 48 hrs (note: this time is variable between 48-72 hrs). 35 ul samples from each well are then transferred to an opaque 96 well plate using a 12 channel pipette. The opaque plates should be covered (using sellophene covers) and stored at ˜20 degrees C. until SEAP assays are performed according to Example 17. The plates containing the remaining treated cells are placed at 4 degrees C. and serve as a source of material for repeating the assay on a specific well if desired.

[1270] As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate Jurkat T cells. Over 30 fold induction is typically observed in the positive control wells.

[1271] The above protocol may be used in the generation of both transient, as well as, stable transfected cells, which would be apparent to those of skill in the art.

Example 14

[1272] High-Throughput Screening Assay Identifying Myeloid Activity

[1273] The following protocol is used to assess myeloid activity by determining whether polypeptides of the invention proliferates and/or differentiates myeloid cells. Myeloid cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 12. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The myeloid cell used in this assay is U937, a pre-monocyte cell line, although TF-1, HL60, or KG1 can be used.

[1274] To transiently transfect U937 cells with the GAS/SEAP/Neo construct produced in Example 12, a DEAE-Dextran method (Kharbanda et. al., 1994, Cell Growth & Differentiation, 5:259-265) is used. First, harvest 2×10e⁷ U937 cells and wash with PBS. The U937 cells are usually grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.

[1275] Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffer containing 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mM NaCl, 5 mM KCl, 375 uM Na₂HPO₄.7H₂O, 1 mM MgCl₂, and 675 uM CaCl₂. Incubate at 37 degrees C. for 45 min.

[1276] Wash the cells with RPM I 1640 medium containing 10% FBS and then resuspend in 10 ml complete medium and incubate at 37 degrees C. for 36 hr.

[1277] The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 400 ug/ml G418 for couple of passages.

[1278] These cells are tested by harvesting 1×10⁸ cells (this is enough for ten 96-well plates assay) and wash with PBS. Suspend the cells in 200 ml above described growth medium, with a final density of 5×10⁵ cells/ml. Plate 200 ul cells per well in the 96-well plate (or 1×10⁵ cells/well).

[1279] Add 50 ul of the supernatant prepared by the protocol described in Example 11. Incubate at 37 degrees C. for 48 to 72 hr. As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate U937 cells. Over 30 fold induction is typically observed in the positive control wells. SEAP assay the supernatant according to the protocol described in Example 17.

Example 15

[1280] High-Throughput Screening Assay Identifying Neuronal Activity.

[1281] When cells undergo differentiation and proliferation, a group of genes are activated through many different signal transduction pathways. One of these genes, EGR1 (early growth response gene 1), is induced in various tissues and cell types upon activation. The promoter of EGR1 is responsible for such induction. Using the EGR1 promoter linked to reporter molecules, activation of cells can be assessed.

[1282] Particularly, the following protocol is used to assess neuronal activity in PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are known to proliferate and/or differentiate by activation with a number of mitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth factor). The EGR1 gene expression is activated during this treatment. Thus, by stably transfecting PC12 cells with a construct containing an EGR promoter linked to SEAP reporter, activation of PC12 cells can be assessed.

[1283] The EGR/SEAP reporter construct can be assembled by the following protocol. The EGR-1 promoter sequence (˜633 to +1)(Sakamoto K et al., Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNA using the following primers: (SEQ ID NO:6) 5′ GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3′ (SEQ ID NO:7) 5′ GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3′

[1284] Using the GAS:SEAP/Neo vector produced in Example 12, EGR1 amplified product can then be inserted into this vector. Linearize the GAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing the GAS/SV40 stuffer. Restrict the EGR1 amplified product with these same enzymes. Ligate the vector and the EGR1 promoter.

[1285] To prepare 96 well-plates for cell culture, two mls of a coating solution (1:30 dilution of collagen type I (Upstate Biotech Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cm plate or 50 ml per well of the 96-well plate, and allowed to air dry for 2 hr.

[1286] PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat. # 12449-78P), 5% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 ug/ml streptomycin on a precoated 10 cm tissue culture dish. One to four split is done every three to four days. Cells are removed from the plates by scraping and resuspended with pipetting up and down for more than 15 times.

[1287] Transfect the EGR/SEAP/Neo construct into PC12 using the Lipofectamine protocol described in Example 11. EGR-SEAP/PC12 stable cells are obtained by growing the cells in 300 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 300 ug/ml G418 for couple of passages.

[1288] To assay for neuronal activity, a 10 cm plate with cells around 70 to 80% confluent is screened by removing the old medium. Wash the cells once with PBS (Phosphate buffered saline). Then starve the cells in low serum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS with antibiotics) overnight.

[1289] The next morning, remove the medium and wash the cells with PBS. Scrape off the cells from the plate, suspend the cells well in 2 ml low serum medium. Count the cell number and add more low serum medium to reach final cell density as 5×10⁵ cells/ml.

[1290] Add 200 ul of the cell suspension to each well of 96-well plate (equivalent to 1×10⁵ cells/well). Add 50 ul supernatant produced by Example 11, 37° C. for 48 to 72 hr. As a positive control, a growth factor known to activate PC12 cells through EGR can be used, such as 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAP is typically seen in the positive control wells. SEAP assay the supernatant according to Example 17.

Example 16

[1291] High-Throughput Screening Assay for T-cell Activity

[1292] NF-KB (Nuclear Factor KB) is a transcription factor activated by a wide variety of agents including the inflammatory cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and by expression of certain viral gene products. As a transcription factor, NF-KB regulates the expression of genes involved in immune cell activation, control of apoptosis (NF-KB appears to shield cells from apoptosis), B and T-cell development, anti-viral and antimicrobial responses, and multiple stress responses.

[1293] In non-stimulated conditions, NF-KB is retained in the cytoplasm with I-KB (Inhibitor KB). However, upon stimulation, I-KB is phosphorylated and degraded, causing NF-KB to shuttle to the nucleus, thereby activating transcription of target genes. Target genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.

[1294] Due to its central role and ability to respond to a range of stimuli, reporter constructs utilizing the NF-KB promoter element are used to screen the supernatants produced in Example 11. Activators or inhibitors of NF-KB would be useful in treating diseases. For example, inhibitors of NF-KB could be used to treat those diseases related to the acute or chronic activation of NF-KB, such as rheumatoid arthritis.

[1295] To construct a vector containing the NF-KB promoter element, a PCR based strategy is employed. The upstream primer contains four tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO: 8), 18 bp of sequence complementary to the 5′ end of the SV40 early promoter sequence, and is flanked with an XhoI site: (SEQ ID NO:9) 5′:GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGG ACTTTCCATCCTGCCATCTCAATTAG:3′

[1296] The downstream primer is complementary to the 3′ end of the SV40 promoter and is flanked with a Hind III site:

[1297] 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO: 4)

[1298] PCR amplification is performed using the SV40 promoter template present in the pB-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI and Hind III and subcloned into BLSK2-. (Stratagene) Sequencing with the T7 and T3 primers confirms the insert contains the following sequence: (SEQ ID NO:10) 5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTT CCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCG CCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGG CTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTG AGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGC AAAAAGCTT:3′

[1299] Next, replace the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40 fragment using XhoI and HindIII. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.

[1300] In order to generate stable mammalian cell lines, the NF-KB/SV40/SEAP cassette is removed from the above NF-KB/SEAP vector using restriction enzymes SalI and NotI, and inserted into a vector containing neomycin resistance. Particularly, the NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing the GFP gene, after restricting pGFP-1 with SalI and NotI.

[1301] Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are created and maintained according to the protocol described in Example 13. Similarly, the method for assaying supernatants with these stable Jurkat T-cells is also described in Example 13. As a positive control, exogenous TNF alpha (0.1,1, 10 ng) is added to wells H9, H10, and H11, with a 5-10 fold activation typically observed.

Example 17

[1302] Assay for SEAP Activity

[1303] As a reporter molecule for the assays described in Examples 13-16, SEAP activity is assayed using the Tropix Phospho-light Kit (Cat. BP-400) according to the following general procedure. The Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction Buffers used below.

[1304] Prime a dispenser with the 2.5× Dilution Buffer and dispense 15 ul of 2.5× dilution buffer into Optiplates containing 35 ul of a supernatant. Seal the plates with a plastic sealer and incubate at 65 degree C. for 30 min. Separate the Optiplates to avoid uneven heating.

[1305] Cool the samples to room temperature for 15 minutes. Empty the dispenser and prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate at room temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see the table below). Add 50 ul Reaction Buffer and incubate at room temperature for 20 minutes. Since the intensity of the chemiluminescent signal is time dependent, and it takes about 10 minutes to read 5 plates on luminometer, one should treat 5 plates at each time and start the second set 10 minutes later.

[1306] Read the relative light unit in the luminometer. Set H12 as blank, and print the results. An increase in chemiluminescence indicates reporter activity. Reaction Buffer Formulation: # of plates Rxn buffer diluent (ml) CSPD (ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 85 4.25 16 90 4.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22 120 6 23 125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.5 29 155 7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 185 9.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41 215 10.75 42 220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47 245 12.25 48 250 12.5 49 255 12.75 50 260 13

Example 18

[1307] High-Throughput Screening Assay Identifying Changes in Small Molecule Concentration and Membrane Permeability

[1308] Binding of a ligand to a receptor is known to alter intracellular levels of small molecules, such as calcium, potassium, sodium, and pH, as well as alter membrane potential. These alterations can be measured in an assay to identify supernatants which bind to receptors of a particular cell. Although the following protocol describes an assay for calcium, this protocol can easily be modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule which is detectable by a fluorescent probe.

[1309] The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) to measure changes in fluorescent molecules (Molecular Probes) that bind small molecules. Clearly, any fluorescent molecule detecting a small molecule can be used instead of the calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.; catalog no. F-14202), used here.

[1310] For adherent cells, seed the cells at 10,000-20,000 cells/well in a Co-star black 96-well plate with clear bottom. The plate is incubated in a CO₂ incubator for 20 hours. The adherent cells are washed two times in Biotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after the final wash.

[1311] A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. To load the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is added to each well. The plate is incubated at 37 degrees C. in a CO₂ incubator for 60 min. The plate is washed four times in the Biotek washer with HBSS leaving 100 ul of buffer.

[1312] For non-adherent cells, the cells are spun down from culture media. Cells are re-suspended to 2-5×10⁶ cells/ml with HBSS in a 50-ml conical tube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is added to each ml of cell suspension. The tube is then placed in a 37 degrees C. water bath for 30-60 min. The cells are washed twice with HBSS, resuspended to 1×10⁶ cells/ml, and dispensed into a microplate, 100 ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate is then washed once in Denley CellWash with 200 ul, followed by an aspiration step to 100 ul final volume.

[1313] For a non-cell based assay, each well contains a fluorescent molecule, such as fluo-4. The supernatant is added to the well, and a change in fluorescence is detected.

[1314] To measure the fluorescence of intracellular calcium, the FLIPR is set for the following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul. Increased emission at 530 nm indicates an extracellular signaling event which has resulted in an increase in the intracellular Ca++ concentration.

Example 19

[1315] High-Throughput Screening Assay Identifying Tyrosine Kinase Activity

[1316] The Protein Tyrosine Kinases (PTK) represent a diverse group of transmembrane and cytoplasmic kinases. Within the Receptor Protein Tyrosine Kinase RPTK) group are receptors for a range of mitogenic and metabolic growth factors including the PDGF, FGF, EGF, NGF, HGF and Insulin receptor subfamilies. In addition there are a large family of RPTKs for which the corresponding ligand is unknown. Ligands for RPTKs include mainly secreted small proteins, but also membrane-bound and extracellular matrix proteins.

[1317] Activation of RPTK by ligands involves ligand-mediated receptor dimerization, resulting in transphosphorylation of the receptor subunits and activation of the cytoplasmic tyrosine kinases. The cytoplasmic tyrosine kinases include receptor associated tyrosine kinases of the src-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked and cytosolic protein tyrosine kinases, such as the Jak family, members of which mediate signal transduction triggered by the cytokine superfamily of receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

[1318] Because of the wide range of known factors capable of stimulating tyrosine kinase activity, the identification of novel human secreted proteins capable of activating tyrosine kinase signal transduction pathways are of interest. Therefore, the following protocol is designed to identify those novel human secreted proteins capable of activating the tyrosine kinase signal transduction pathways.

[1319] Seed target cells (e.g., primary keratinocytes) at a density of approximately 25,000 cells per well in a 96 well Loprodyne Silent Screen Plates purchased from Nalge Nunc (Naperville, Ill.). The plates are sterilized with two 30 minute rinses with 100% ethanol, rinsed with water and dried overnight. Some plates are coated for 2 hr with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can be purchased from Sigma Chemicals (St. Louis, Mo.) or 10% Matrigel purchased from Becton Dickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at 4 degree C. Cell growth on these plates is assayed by seeding 5,000 cells/well in growth medium and indirect quantitation of cell number through use of alamarBlue as described by the manufacturer Alamar Biosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers #3071 from Becton Dickinson (Bedford, Mass,) are used to cover the Loprodyne Silent Screen Plates. Falcon Microtest III cell culture plates can also be used in some proliferation experiments.

[1320] To prepare extracts, A431 cells are seeded onto the nylon membranes of Loprodyne plates (20,000/200 ml/well) and cultured overnight in complete medium. Cells are quiesced by incubation in serum-free basal medium for 24 hr. After 5-20 minutes treatment with EGF (60 ng/ml) or 50 ul of the supernatant produced in Example 11, the medium was removed and 100 ml of extraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4, 2 mM Na4P207 and a cocktail of protease inhibitors (#1836170) obtained from Boeheringer Mannheim (Indianapolis, Ind.) is added to each well and the plate is shaken on a rotating shaker for 5 minutes at 4 degrees C. The plate is then placed in a vacuum transfer manifold and the extract filtered through the 0.45 mm membrane bottoms of each well using house vacuum. Extracts are collected in a 96-well catch/assay plate in the bottom of the vacuum manifold and immediately placed on ice. To obtain extracts clarified by centrifugation, the content of each well, after detergent solubilization for 5 minutes, is removed and centrifuged for 15 minutes at 4 degrees C at 16,000×g.

[1321] Test the filtered extracts for levels of tyrosine kinase activity. Although many methods of detecting tyrosine kinase activity are known, one method is described here.

[1322] Generally, the tyrosine kinase activity of a supernatant is evaluated by determining its ability to phosphorylate a tyrosine residue on a specific substrate (a biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for a range of tyrosine kinases and are available from Boehringer Mannheim.

[1323] The tyrosine kinase reaction is set up by adding the following components in order. First, add 10 ul of 5 uM Biotinylated Peptide, then 10 ul ATP/Mg₂₊ (5 mM ATP/50mM MgCl₂), then 10 ul of 5× Assay Buffer (40 mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100 mM MgCl₂, 5 mM MnCl₂, 0.5 mg/ml BSA), then 5 ul of Sodium Vanadate(1 mM), and then 5 ul of water. Mix the components gently and preincubate the reaction mix at 30 degrees C. for 2 min. Initial the reaction by adding 10 ul of the control enzyme or the filtered supernatant.

[1324] The tyrosine kinase assay reaction is then terminated by adding 10 ul of 120 mm EDTA and place the reactions on ice.

[1325] Tyrosine kinase activity is determined by transferring 50 ul aliquot of reaction mixture to a microtiter plate (MTP) module and incubating at 37 degrees C. for 20 min. This allows the streptavadin coated 96 well plate to associate with the biotinylated peptide. Wash the MTP module with 300 ul/well of PBS four times. Next add 75 ul of anti-phospotyrosine antibody conjugated to horse radish peroxidase(anti-P-Tyr-POD(0.5 u/ml)) to each well and incubate at 37 degrees C. for one hour. Wash the well as above.

[1326] Next add 100 ul of peroxidase substrate solution (Boehringer Mannheim) and incubate at room temperature for at least 5 mins (up to 30 min). Measure the absorbance of the sample at 405 nm by using ELISA reader. The level of bound peroxidase activity is quantitated using an ELISA reader and reflects the level of tyrosine kinase activity.

Example 20

[1327] High-Throughput Screening Assay Identifying Phosphorylation Activity

[1328] As a potential alternative and/or compliment to the assay of protein tyrosine kinase activity described in Example 19, an assay which detects activation (phosphorylation) of major intracellular signal transduction intermediates can also be used. For example, as described below one particular assay can detect tyrosine phosphorylation of the Erk-1 and Erk-2 kinases. However, phosphorylation of other molecules, such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine, phosphotyrosine, or phosphothreonine molecule, can be detected by substituting these molecules for Erk-1 or Erk-2 in the following assay.

[1329] Specifically, assay plates are made by coating the wells of a 96-well ELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at room temp, (RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBS for 1 hr at RT. The protein G plates are then treated with 2 commercial monoclonal antibodies (100 ng/well) against Erk-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology). (To detect other molecules, this step can easily be modified by substituting a monoclonal antibody detecting any of the above described molecules.) After 3-5 rinses with PBS, the plates are stored at 4 degrees C. until use.

[1330] A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplate and cultured overnight in growth medium. The cells are then starved for 48 hr in basal medium (DMEM) and then treated with EGF (6 ng/well) or 50 ul of the supernatants obtained in Example 11 for 5-20 minutes. The cells are then solubilized and extracts filtered directly into the assay plate.

[1331] After incubation with the extract for 1 hr at RT, the wells are again rinsed. As a positive control, a commercial preparation of MAP kinase (10 ng/well) is used in place of A431 extract. Plates are then treated with a commercial polyclonal (rabbit) antibody (1 ug/ml) which specifically recognizes the phosphorylated epitope of the Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is biotinylated by standard procedures. The bound polyclonal antibody is then quantitated by successive incubations with Europium-streptavidin and Europium fluorescence enhancing reagent in the Wallac DELFIA instrument (time-resolved fluorescence). An increased fluorescent signal over background indicates a phosphorylation.

Example 21

[1332] Method of Determining Alterations in a Gene Corresponding to a Polynucleotide

[1333] RNA isolated from entire families or individual patients presenting with a phenotype of interest (such as a disease) is be isolated. cDNA is then generated from these RNA samples using protocols known in the art. (See, Sambrook.) The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO: X. Suggested PCR conditions consist of 35 cycles at 95 degrees C. for 30 seconds; 60-120 seconds at 52-58 degrees C.; and 60-120 seconds at 70 degrees C., using buffer solutions described in Sidransky et al., Science 252:706 (1991).

[1334] PCR products are then sequenced using primers labeled at their 5′ end with T4 polynucleotide kinase, employing SequiTherm Polymerase. (Epicentre Technologies). The intron-exon borders of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations is then cloned and sequenced to validate the results of the direct sequencing.

[1335] PCR products is cloned into T-tailed vectors as described in Holton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals.

[1336] Genomic rearrangements are also observed as a method of determining alterations in a gene corresponding to a polynucleotide. Genomic clones isolated according to Example 2 are nick-translated with digoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISH performed as described in Johnson et al., Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeled probe is carried out using a vast excess of human cot-1 DNA for specific hybridization to the corresponding genomic locus.

[1337] Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C- and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, Vt.) in combination with a cooled charge-coupled device camera (Photometrics, Tucson, Ariz.) and variable excitation wavelength filters. (Johnson et al., Genet. Anal. Tech. Appl., 8:75 (1991).) Image collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Corporation, Durham, N.C.) Chromosome alterations of the genomic region hybridized by the probe are identified as insertions, deletions, and translocations. These alterations are used as a diagnostic marker for an associated disease.

Example 22

[1338] Method of Detecting Abnormal Levels of a Polypeptide in a Biological Sample

[1339] A polypeptide of the present invention can be detected in a biological sample, and if an increased or decreased level of the polypeptide is detected, this polypeptide is a marker for a particular phenotype. Methods of detection are numerous, and thus, it is understood that one skilled in the art can modify the following assay to fit their particular needs.

[1340] For example, antibody-sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies, at a final concentration of 0.2 to 10 ug/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described in Example 10. The wells are blocked so that non-specific binding of the polypeptide to the well is reduced.

[1341] The coated wells are then incubated for >2 hours at RT with a sample containing the polypeptide. Preferably, serial dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled water to remove unbounded polypeptide.

[1342] Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at a concentration of 25-400 ng, is added and incubated for 2 hours at room temperature. The plates are again washed three times with deionized or distilled water to remove unbounded conjugate.

[1343] Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate 1 hour at room temperature. Measure the reaction by a microtiter plate reader. Prepare a standard curve, using serial dilutions of a control sample, and plot polypeptide concentration on the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear scale). Interpolate the concentration of the polypeptide in the sample using the standard curve.

Example 23

[1344] Formulation

[1345] The invention also provides methods of treatment and/or prevention of diseases or disorders (such as, for example, any one or more of the diseases or disorders disclosed herein) by administration to a subject of an effective amount of a Therapeutic. By therapeutic is meant polynucleotides or polypeptides of the invention (including fragments and variants), agonists or antagonists thereof, and/or antibodies thereto, in combination with a pharmaceutically acceptable carrier type (e.g., a sterile carrier).

[1346] The Therapeutic will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the Therapeutic alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations.

[1347] As a general proposition, the total pharmaceutically effective amount of the Therapeutic administered parenterally per dose will be in the range of about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the Therapeutic is typically administered at a dose rate of about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.

[1348] Therapeutics can be are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[1349] Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[1350] Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).

[1351] Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).

[1352] In a preferred embodiment, Neutrokine-alpha and/or Neutrokine-alphaSV compositions of the invention are formulated in a biodegradable, polymeric drug delivery system, for example as described in U.S. Pat. Nos. 4,938,763; 5,278,201; 5,278,202; 5,324,519; 5,340,849; and 5,487,897 and in International Publication Numbers WO01/35929, WO/0024374, and WO/0006117 which are hereby incorporated by reference in their entirety. In specific preferred embodiments the Neutrokine-alpha and/or Neutrokine-alphaSV compositions of the invention are formulated using the ATRIGEL® Biodegradable System of Atrix Laboratories, Inc. (Fort Collins, Colo.).

[1353] Examples of biodegradable polymers which can be used in the formulation of Neutrokine-alpha and/or Neutrokine-alphaSV compositions, include but are not limited to, polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(amino acids), poly(methyl vinyl ether), poly(maleic anhydride), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, chitin, chitosan, and copolymers, terpolymers, or combinations or mixtures of the above materials. The preferred polymers are those that have a lower degree of crystallization and are more hydrophobic. These polymers and copolymers are more soluble in the biocompatible solvents than the highly crystalline polymers such as polyglycolide and chitin which also have a high degree of hydrogen-bonding. Preferred materials with the desired solubility parameters are the polylactides, polycaprolactones, and copolymers of these with glycolide in which there are more amorphous regions to enhance solubility. In specific preferred embodiments, the biodegradable polymers which can be used in the formulation of Neutrokine-alpha and/or Neutrokine-alphaSV compositions are poly(lactide-co-glycolides). Polymer properties such as molecular weight, hydrophobicity, and lactide/glycolide ratio may be modified to obtain the desired drug Neutrokine-alpha and/or Neutrokine-alphaSV release profile (See, e.g., Ravivarapu et al., Journal of Pharmaceutical Sciences 89:732-741 (2000), which is hereby incorporated by refernce in its entirety).

[1354] It is also preferred that the solvent for the biodegradable polymer be non-toxic, water miscible, and otherwise biocompatible. Examples of such solvents include, but are not limted to, N-methyl-2-pyrrolidone, 2-pyrrolidone, C2 to C6 alkanols, C1 to C15 alchohols, dils, triols, and tetraols such as ethanol, glycerine propylene glycol, butanol; C3 to C15 alkyl ketones such as acetone, diethyl ketone and methyl ethyl ketone; C3 to C15 esters such as methyl acetate, ethyl acetate, ethyl lactate; alkyl ketones such as methyl ethyl ketone, C1 to C15 amides such as dimethylformamide, dimethylacetamide and caprolactam; C3 to C20 ethers such as tetrahydrofuran, or solketal; tweens, triacetin, propylene carbonate, decylmethylsulfoxide, dimethyl sulfoxide, oleic acid, 1-dodecylazacycloheptan-2-one, Other preferred solvents are benzyl alchohol, benzyl benzoate, dipropylene glycol, tributyrin, ethyl oleate, glycerin, glycofural, isopropyl myristate, isopropyl palmitate, oleic acid, polyethylene glycol, propylene carbonate, and triethyl citrate. The most preferred solvents are

[1355] N-methyl-2-pyrrolidone, 2-pyrrolidone, dimethyl sulfoxide, triacetin, and propylene carbonate because of the solvating ability and their compatibility.

[1356] Additionally, formulations comprising Neutrokine-alpha and/or Neutrokine-alphaSV compositions and a biodegradable polymer may also include release-rate modification agents and/or pore-forming agents. Examples of release-rate modification agents include, but are not limited to, fatty acids, triglycerides, other like hydrophobic compounds, organic solvents, plasticizing compounds and hydrophilic compounds. Suitable release rate modification agents include, for example, esters of mono-, di-, and tricarboxylic acids, such as 2-ethoxyethyl acetate, methyl acetate, ethyl acetate, diethyl phthalate, dimethyl phthalate, dibutyl phthalate, dimethyl adipate, dimethyl succinate, dimethyl oxalate, dimethyl citrate, triethyl citrate, acetyl tributyl citrate, acetyl triethyl citrate, glycerol triacetate, di(n-butyl) sebecate, and the like; polyhydroxy alcohols, such as propylene glycol, polyethylene glycol, glycerin, sorbitol, and the like; fatty acids; triesters of glycerol, such as triglycerides, epoxidized soybean oil, and other epoxidized vegetable oils; sterols, such as cholesterol; alcohols, such as C.sub.6 -C.sub.12 alkanols, 2-ethoxyethanol, and the like. The release rate modification agent may be used singly or in combination with other such agents. Suitable combinations of release rate modification agents include, but are not limited to, glycerin/propylene glycol, sorbitol/glycerine, ethylene oxide/propylene oxide, butylene glycol/adipic acid, and the like. Preferred release rate modification agents include, but are not limited to, dimethyl citrate, triethyl citrate, ethyl heptanoate, glycerin, and hexanediol. Suitable pore-forming agents that may be used in the polymer composition include, but are not limited to, sugars such as sucrose and dextrose, salts such as sodium chloride and sodium carbonate, polymers such as hydroxylpropylcellulose, carboxymethylcellulose, polyethylene glycol, and polyvinylpyrrolidone. Solid crystals that will provide a defined pore size, such as salt or sugar, are preferred.

[1357] In specific preferred embodiments the Neutrokine-alpha and/or Neutrokine-alphaSV compositions of the invention are formulated using the BEMA™ BioErodible Mucoadhesive System, MCA™ MucoCutaneous Absorption System, SMP™ Solvent MicroParticle System, or BCP™ BioCompatible Polymer System of Atrix Laboratories, Inc. (Fort Collins, Colo.).

[1358] Sustained-release Therapeutics also include liposomally entrapped Therapeutics of the invention (see generally, Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317 -327 and 353-365 (1989)). Liposomes containing the Therapeutic are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); E P 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal Therapeutic.

[1359] In yet an additional embodiment, the Therapeutics of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).

[1360] Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

[1361] For parenteral administration, in one embodiment, the Therapeutic is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.

[1362] Generally, the formulations are prepared by contacting the Therapeutic uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.

[1363] The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

[1364] The Therapeutic is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.

[1365] Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutics generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[1366] Therapeutics ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous Therapeutic solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized Therapeutic using bacteriostatic Water-for-Injection.

[1367] The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the Therapeutics of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the Therapeutics may be employed in conjunction with other therapeutic compounds.

[1368] The Therapeutics of the invention may be administered alone or in combination with adjuvants. Adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG (e.g., THERACYS®), MPL and nonviable prepartions of Corynebacterium parvum. In a specific embodiment, Therapeutics of the invention are administered in combination with alum. In another specific embodiment, Therapeutics of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the Therapeutics of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

[1369] The Therapeutics of the invention may be administered alone or in combination with other therapeutic agents. Therapeutic agents that may be administered in combination with the Therapeutics of the invention, include but not limited to, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, and/or therapeutic treatments described below. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

[1370] In certain embodiments, Therapeutics of the invention are administered in combination with antiretroviral agents, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and/or protease inhibitors (PIs). NRTIs that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC), ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™ (zidovudine/lamivudine). NNRTIs that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, VIRAMUNE™ (nevirapine), RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, CRIXIVAN™ (indinavir), NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with Therapeutics of the invention to treat AIDS and/or to prevent or treat HIV infection.

[1371] Additional NRTIs include LODENOSINE™ (F-ddA; an acid-stable adenosine NRTI; Triangle/Abbott; COVIRACIL™ (emtricitabine/PTC; structurally related to lamivudine (3TC) but with 3-to 10-fold greater activity in vitro; Triangle/Abbott); dOTC (BCH-10652, also structurally related to lamivudine but retains activity against a substantial proportion of lamivudine-resistant isolates; Biochem Pharma); Adefovir (refused approval for anti-HIV therapy by FDA; Gilead Sciences); PREVEON® (Adefovir Dipivoxil, the active prodrug of adefovir; its active form is PMEA-pp); TENOFOVIR™ (bis-POC PMPA, a PMPA prodrug; Gilead); DAPD/DXG (active metabolite of DAPD; Triangle/Abbott); D-D4FC (related to 3TC, with activity against AZT/3TC-resistant virus); GW420867X (Glaxo Wellcome); ZIAGEN™ (abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3′ azido-2′,3′-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl (SATE)-bearing prodrug forms of β-L-FD4C and β-L-FddC (WO 98/17281).

[1372] Additional NNRTIs include COACTINON™ (Emivirine/MKC-442, potent NNRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINE™ (AG-1549/S-1153, a next generation NNRTI with activity against viruses containing the K103N mutation; Agouron); PNU-142721 (has 20-to 50-fold greater activity than its predecessor delavirdine and is active against K103N mutants; Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generation derivatives of efavirenz, designed to be active against viruses with the K103N mutation; DuPont); GW-420867X (has 25-fold greater activity than HBY097 and is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A (naturally occurring agent from the latex tree; active against viruses containing either or both the Y181C and K103N mutations); and Propolis (WO 99/49830).

[1373] Additional protease inhibitors include LOPINAVIR™ (ABT378/r; Abbott Laboratories); BMS-232632 (an azapeptide; Bristol-Myres Squibb); TIPRANAVIR™ (PNU-140690, a non-peptic dihydropyrone; Pharmacia & Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide; Bristol-Myers Squibb); L-756,423 (an indinavir analog; Merck); DMP-450 (a cyclic urea compound; Avid & DuPont); AG-1776 (a peptidomimetic with in vitro activity against protease inhibitor-resistant viruses; Agouron); VX-175/GW-433908 (phosphate prodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755 (Ciba); and AGENERASE™ (amprenavir; Glaxo Wellcome Inc.).

[1374] Additional antiretroviral agents include fusion inhibitors/gp41 binders. Fusion inhibitors/gp41 binders include T-20 (a peptide from residues 643-678 of the HIV gp41 transmembrane protein ectodomain which binds to gp41 in its resting state and prevents transformation to the fusogenic state; Trimeris) and T-1249 (a second-generation fusion inhibitor; Trimeris).

[1375] Additional antiretroviral agents include fusion inhibitors/chemokine receptor antagonists. Fusion inhibitors/chemokine receptor antagonists include CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX40-4C (a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and the T22 analogs T134 and T140; CCR5 antagonists such as RANTES (9-68), AOP-RANTES, NNY-RANTES, and TAK-779; and CCR5/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Also included are CCR2B, CCR3, and CCR6 antagonists. Chemokine recpetor agonists such as RANTES, SDF-1, MIP-1α, MIP-1β, etc., may also inhibit fusion.

[1376] Additional antiretroviral agents include integrase inhibitors. Integrase inhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (a dicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and related anthraquinones; ZINTEVIR™ (AR 177, an oligonucleotide that probably acts at cell surface rather than being a true integrase inhibitor; Arondex); and naphthols such as those disclosed in WO 98/50347.

[1377] Additional antiretroviral agents include hydroxyurea-like compunds such as BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst); ribonucleotide reductase inhibitors such as DIDOX™ (Molecules for Health); inosine monophosphate dehydrogenase (IMPDH) inhibitors sucha as VX-497 (Vertex); and mycopholic acids such as CellCept (mycophenolate mofetil; Roche).

[1378] Additional antiretroviral agents include inhibitors of viral integrase, inhibitors of viral genome nuclear translocation such as arylene bis(methylketone) compounds; inhibitors of HIV entry such as AOP-RANTES, NNY-RANTES, RANTES-IgG fusion protein, soluble complexes of RANTES and glycosaminoglycans (GAG), and AMD-3 100; nucleocapsid zinc finger inhibitors such as dithiane compounds; targets of HIV Tat and Rev; and pharmacoenhancers such as ABT-378.

[1379] Other antiretroviral therapies and adjunct therapies include cytokines and lymphokines such as MIP-1α, MIP-1β, SDF-1α, IL-2, PROLEUKIN™ (aldesleukin/L2-7001; Chiron), IL-4, IL-10, IL-12, and IL-13; interferons such as IFN-α2a; antagonists of TNFs, NFκB, GM-CSF, M-CSF, and IL-10; agents that modulate immune activation such as cyclosporin and prednisone; vaccines such as Remune™ (HIV Immunogen), APL 400-003 (Apollon), recombinant gp120 and fragments, bivalent (B/E) recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2 rgp120, gp l20/soluble CD4 complex, Delta JR-FL protein, branched synthetic peptide derived from discontinuous gp120 C3/C4 domain, fusion-competent immunogens, and Gag, Pol, Nef, and Tat vaccines; gene-based therapies such as genetic suppressor elements (GSEs; WO 98/54366), and intrakines (genetically modified CC chemokines targetted to the ER to block surface expression of newly synthesized CCR5 (Yang et al., PNAS 94:11567-72 (1997); Chen et al., Nat. Med. 3:1110-16 (1997)); antibodies such as the anti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9, PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA-T4, the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies, anti-TNF-α antibodies, and monoclonal antibody 33A; aryl hydrocarbon (AH) receptor agonists and antagonists such as TCDD, 3,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′-tetrachlorobiphenyl, and α-naphthoflavone (WO 98/30213); and antioxidants such as γ-L-glutamyl-L-cysteine ethyl ester (γ-GCE; WO 99/56764).

[1380] In a further embodiment, the Therapeutics of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the Therapeutics of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.

[1381] In other embodiments, Therapeutics of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™, ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™, CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™, FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™, PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™ (sargramostim/GM-CSF). In a specific embodiment, Therapeutics of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/or ATOVAQUONE™ to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ to prophylactically treat or prevent an opportunistic Mycobacterium avium complex infection. In another specific embodiment, Therapeutics of the invention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, Therapeutics of the invention are used in any combination with GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylactically treat or prevent an opportunistic cytomegalovirus infection. In another specific embodiment, Therapeutics of the invention are used in any combination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, Therapeutics of the invention are used in any combination with PYRIMETHAMINE™ and/or LEUCOVORIN™ to prophylactically treat or prevent an opportunistic Toxoplasma gondii infection. In another specific embodiment, Therapeutics of the invention are used in any combination with LEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent an opportunistic bacterial infection.

[1382] In a further embodiment, the Therapeutics of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the Therapeutics of the invention include, but are not limited to, amoxicillin, beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rapamycin, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.

[1383] In other embodiments, Therapeutics of the invention are administered in combination with immunosuppressive agents. Immunosuppressive agents that may be administered in combination with the Therapeutics of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells. Other immunosuppressive agents that may be administered in combination with the Therapeutics of the invention include, but are not limited to, prednisolone, methotrexate, thalidomide, methoxsalen, rapamycin, leflunomide, mizoribine (BREDININ™), brequinar, deoxyspergualin, and azaspirane (SKF 105685), ORTHOCLONE OKT® 3 (muromonab-CD3), SANDIMMUNE™, NEORAL™, SANGDYA™ (cyclosporine), PROGRAF® (FK506, tacrolimus), CELLCEPT® (mycophenolate motefil, of which the active metabolite is mycophenolic acid), IMURAN™ (azathioprine), glucocorticosteroids, adrenocortical steroids such as DELTASONE™ (prednisone) and HYDELTRASOL™ (prednisolone), FOLEX™ and MEXATE™ (methotrxate), OXSORALEN-ULTRA™ (methoxsalen) and RAPAMUNE™ (sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.

[1384] In an additional embodiment, Therapeutics of the invention are administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the Therapeutics of the invention include, but not limited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, ATGAM™ (antithymocyte glubulin), and GAMIMUNE™. In a specific embodiment, Therapeutics of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).

[1385] In certain embodiments, the Therapeutics of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the Therapeutics of the invention include, but are not limited to, corticosteroids (e.g. betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), nonsteroidal anti-inflammatory drugs (e.g., diclofenac, diflunisal, etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic acid, and tolmetin.), as well as antihistamines, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.

[1386] In an additional embodiment, the compositions of the invention are administered alone or in combination with an anti-angiogenic agent. Anti-angiogenic agents that may be administered with the compositions of the invention include, but are not limited to, Angiostatin (Entremed, Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Mass.), anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel (Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.

[1387] Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.

[1388] Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono-and trihydrates.

[1389] Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.

[1390] A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include, but are not limited to, platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, (1991)); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, (1992)); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, (1992)); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, (1990)); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, (1987)); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4): 1659-1664, (1987)); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl4-chloroanthronilic acid disodium or “CCA”; (Takeuchi et al., Agents Actions 36:312-316, (1992)); and metalloproteinase inhibitors such as BB94.

[1391] Additional anti-angiogenic factors that may also be utilized within the context of the present invention include Thalidomide, (Celgene, Warren, N.J.); Angiostatic steroid; AGM-1470 (H. Brem and J. Folkman J Pediatr. Surg. 28:445-51 (1993)); an integrin alpha v beta 3 antagonist (C. Storgard et al., J Clin. Invest. 103:47-54 (1999)); carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National Cancer Institute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXiGENE, Boston, Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.); TNP-470, (Tap Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca (London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251 (PKC 412); CM101; Dexrazoxane (ICRF1 87); DMXAA; Endostatin; Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide (Somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat (AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex); Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and 5-Fluorouracil.

[1392] Anti-angiogenic agents that may be administed in combination with the compounds of the invention may work through a variety of mechanisms including, but not limited to, inhibiting proteolysis of the extracellular matrix, blocking the function of endothelial cell-extracellular matrix adhesion molecules, by antagonizing the function of angiogenesis inducers such as growth factors, and inhibiting integrin receptors expressed on proliferating endothelial cells. Examples of anti-angiogenic inhibitors that interfere with extracellular matrix proteolysis and which may be administered in combination with the compositons of the invention include, but are not lmited to, AG-3340 (Agouron, La Jolla, Calif.), BAY-12-9566 (Bayer, West Haven, Conn.), BMS-275291 (Bristol Myers Squibb, Princeton, N.J.), CGS-27032A (Novartis, East Hanover, N.J.), Marimastat (British Biotech, Oxford, UK), and Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenic inhibitors that act by blocking the function of endothelial cell-extracellular matrix adhesion molecules and which may be administered in combination with the compositons of the invention include, but are not lmited to, EMD-121974 (Merck KcgaA Darmstadt, Germany) and Vitaxin (Ixsys, La Jolla, Calif./Medimmune, Gaithersburg, Md.). Examples of anti-angiogenic agents that act by directly antagonizing or inhibiting angiogenesis inducers and which may be administered in combination with the compositons of the invention include, but are not lmited to, Angiozyme (Ribozyme, Boulder, Colo.), Anti-VEGF antibody (Genentech, S. San Francisco, Calif.), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. San Francisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, N.J.), and SU-6668 (Sugen). Other anti-angiogenic agents act to indirectly inhibit angiogenesis. Examples of indirect inhibitors of angiogenesis which may be administered in combination with the compositons of the invention include, but are not limited to, IM-862 (Cytran, Kirkland, Wash.), Interferon-alpha, IL-12 (Roche, Nutley, N.J.), and Pentosan polysulfate (Georgetown University, Washington, D.C.).

[1393] In particular embodiments, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of an autoimmune disease, such as for example, an autoimmune disease described herein.

[1394] In a particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of arthritis. In a more particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of rheumatoid arthritis.

[1395] In another embodiment, the polynucleotides encoding a polypeptide of the present invention are administered in combination with an angiogenic protein, or polynucleotides encoding an angiogenic protein. Examples of angiogenic proteins that may be administered with the compositions of the invention include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.

[1396] In additional embodiments, compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the Therapeutics of the invention include, but are not limited to alkylating agents such as nitrogen mustards (for example, Mechlorethamine, cyclophosphamide, Cyclophosphamide Ifosfamide, Melphalan (L-sarcolysin), and Chlorambucil), ethylenimines and methylmelamines (for example, Hexamethylmelamine and Thiotepa), alkyl sulfonates (for example, Busulfan), nitrosoureas (for example, Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl-CCNU), and Streptozocin (streptozotocin)), triazenes (for example, Dacarbazine (DTIC; dimethyltriazenoimidazolecarboxamide)), folic acid analogs (for example, Methotrexate (amethopterin)), pyrimidine analogs (for example, Fluorouacil (5-fluorouracil; 5-FU), Floxuridine (fluorodeoxyuridine; FudR), and Cytarabine (cytosine arabinoside)), purine analogs and related inhibitors (for example, Mercaptopurine (6-mercaptopurine; 6-MP), Thioguanine (6-thioguanine; TG), and Pentostatin (2′-deoxycoformycin)), vinca alkaloids (for example, Vinblastine (VLB, vinblastine sulfate)) and Vincristine (vincristine sulfate)), epipodophyllotoxins (for example, Etoposide and Teniposide), antibiotics (for example, Dactinomycin (actinomycin D), Daunorubicin (daunomycin; rubidomycin), Doxorubicin, Bleomycin, Plicamycin (mithramycin), and Mitomycin (mitomycin C), enzymes (for example, L-Asparaginase), biological response modifiers (for example, Interferon-alpha and interferon-alpha-2b), platinum coordination compounds (for example, Cisplatin (cis-DDP) and Carboplatin), anthracenedione (Mitoxantrone), substituted ureas (for example, Hydroxyurea), methylhydrazine derivatives (for example, Procarbazine (N-methylhydrazine; MIH), adrenocorticosteroids (for example, Prednisone), progestins (for example, Hydroxyprogesterone caproate, Medroxyprogesterone, Medroxyprogesterone acetate, and Megestrol acetate), estrogens (for example, Diethylstilbestrol (DES), Diethylstilbestrol diphosphate, Estradiol, and Ethinyl estradiol), antiestrogens (for example, Tamoxifen), androgens (Testosterone proprionate, and Fluoxymesterone), antiandrogens (for example, Flutamide), gonadotropin-releasing horomone analogs (for example, Leuprolide), other hormones and hormone analogs (for example, methyltestosterone, estramustine, estramustine phosphate sodium, chlorotrianisene, and testolactone), and others (for example, dicarbazine, glutamic acid, and mitotane).

[1397] In one embodiment, the compositions of the invention are administered in combination with one or more of the following drugs: infliximab (also known as Remicade™ Centocor, Inc.), Trocade (Roche, RO-32-3555), Leflunomide (also known as Arava™ from Hoechst Marion Roussel), Kineret™ (an IL-1 Receptor antagonist also known as Anakinra from Amgen, Inc.)

[1398] In a specific embodiment, compositions of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or combination of one or more of the components of CHOP. In one embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies, human monoclonal anti-CD20 antibodies. In another embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies and CHOP, or anti-CD20 antibodies and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with Rituximab. In a further embodiment, compositions of the invention are administered with Rituximab and CHOP, or Rituximab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with tositumomab. In a further embodiment, compositions of the invention are administered with tositumomab and CHOP, or tositumomab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. The anti-CD20 antibodies may optionally be associated with radioisotopes, toxins or cytotoxic prodrugs.

[1399] In another specific embodiment, the compositions of the invention are administered in combination Zevalin™. In a further embodiment, compositions of the invention are administered with Zevalin™ and CHOP, or Zevalin™ and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. Zevalin™ may be associated with one or more radisotopes. Particularly preferred isotopes are ⁹⁰Y and ¹¹¹In.

[1400] In an additional embodiment, the Therapeutics of the invention are administered in combination with cytokines. Cytokines that may be administered with the Therapeutics of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment, Therapeutics of the invention may be administered with any interleukin, including, but not limited to, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

[1401] In one embodiment, the Therapeutics of the invention are administered in combination with members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the Therapeutics of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), endokine-alpha (International Publication No. WO 98/07880), OPG, and neutrokine-alpha (International Publication No. WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TRANK, TR9 (International Publication No. WO 98/56892),TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.

[1402] In an additional embodiment, the Therapeutics of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the Therapeutics of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-6821 10; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PlGF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (PlGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268-(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO 96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DE19639601. The above mentioned references are herein incorporated by reference in their entireties.

[1403] In an additional embodiment, the Therapeutics of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the Therapeutics of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.

[1404] In an additional embodiment, the Therapeutics of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the Therapeutics of the invention include, but are not limited to, granulocyte macrophage colony stimulating factor (GM-CSF) (sargramostim, LEUKINE™, PROKINE™), granulocyte colony stimulating factor (G-CSF) (filgrastim, NEUPOGEN™), macrophage colony stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin alfa, EPOGEN™, PROCRIT™), stem cell factor (SCF, c-kit ligand, steel factor), megakaryocyte colony stimulating factor, PIXY321 (a GMCSF/IL-3 fusion protein), interleukins, especially any one or more of IL-1 through IL-12, interferon-gamma, or thrombopoietin.

[1405] In certain embodiments, Therapeutics of the present invention are administered in combination with adrenergic blockers, such as, for example, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, labetalol, metoprolol, nadolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol, and timolol.

[1406] In another embodiment, the Therapeutics of the invention are administered in combination with an antiarrhythmic drug (e.g., adenosine, amidoarone, bretylium, digitalis, digoxin, digitoxin, diliazem, disopyramide, esmolol, flecainide, lidocaine, mexiletine, moricizine, phenytoin, procainamide, N-acetyl procainamide, propafenone, propranolol, quinidine, sotalol, tocainide, and verapamil).

[1407] In another embodiment, the Therapeutics of the invention are administered in combination with diuretic agents, such as carbonic anhydrase-inhibiting agents (e.g., acetazolamide, dichlorphenamide, and methazolamide), osmotic diuretics (e.g., glycerin, isosorbide, mannitol, and urea), diuretics that inhibit Na⁺-K⁺-2Cl⁻ symport (e.g., furosemide, bumetanide, azosemide, piretanide, tripamide, ethacrynic acid, muzolimine, and torsemide), thiazide and thiazide-like diuretics (e.g., bendroflumethiazide, benzthiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, polythiazide, trichormethiazide, chlorthalidone, indapamide, metolazone, and quinethazone), potassium sparing diuretics (e.g., amiloride and triamterene), and mineralcorticoid receptor antagonists (e.g., spironolactone, canrenone, and potassium canrenoate).

[1408] In one embodiment, the Therapeutics of the invention are administered in combination with treatments for endocrine and/or hormone imbalance disorders. Treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, ¹²⁷I, radioactive isotopes of iodine such as ¹³¹I and ¹²³I; recombinant growth hormone, such as HUMATROPE™ (recombinant somatropin); growth hormone analogs such as PROTROPIN™ (somatrem); dopamine agonists such as PARLODEL™ (bromocriptine); somatostatin analogs such as SANDOSTATIN™ (octreotide); gonadotropin preparations such as PREGNYL™, A.P.L.™ and PROFASI™ (chorionic gonadotropin (CG)), PERGONAL™ (menotropins), and METRODIN™ (urofollitropin (uFSH)); synthetic human gonadotropin releasing hormone preparations such as FACTREL™ and LUTREPULSE™ (gonadorelin hydrochloride); synthetic gonadotropin agonists such as LUPRON™ (leuprolide acetate), SUPPRELIN™ (histrelin acetate), SYNAREL™ (nafarelin acetate), and ZOLADEX™ (goserelin acetate); synthetic preparations of thyrotropin-releasing hormone such as RELEFACT TRH™ and THYPINONE™ (protirelin); recombinant human TSH such as THYROGEN™; synthetic preparations of the sodium salts of the natural isomers of thyroid hormones such as L-T₄™, SYNTHROID™ and LEVOTHROID™ (levothyroxine sodium), L-T₃™, CYTOMEL™ and TRIOSTAT™ (liothyroine sodium), and THYROLAR™ (liotrix); antithyroid compounds such as 6-n-propylthiouracil (propylthiouracil), 1-methyl-2-mercaptoimidazole and TAPAZOLE™ (methimazole), NEO-MERCAZOLE™ (carbimazole); beta-adrenergic receptor antagonists such as propranolol and esmolol; Ca²⁺ channel blockers; dexamethasone and iodinated radiological contrast agents such as TELEPAQUE™ (iopanoic acid) and ORAGRAFIN™ (sodium ipodate).

[1409] Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, estrogens or congugated estrogens such as ESTRACE™ (estradiol), ESTINYL™ (ethinyl estradiol), PREMARIN™, ESTRATAB™, ORTHO-EST™, OGEN™ and estropipate (estrone), ESTROVIS™ (quinestrol), ESTRADERM™ (estradiol), DELESTROGEN™ and VALERGEN™ (estradiol valerate), DEPO-ESTRADIOL CYPIONATE™ and ESTROJECT LA™ (estradiol cypionate); antiestrogens such as NOLVADEX™ (tamoxifen), SEROPHENE™ and CLOMID™ (clomiphene); progestins such as DURALUTIN™ (hydroxyprogesterone caproate), MPA™ and DEPO-PROVERA™ (medroxyprogesterone acetate), PROVERA™ and CYCRIN™ (MPA), MEGACE™ (megestrol acetate), NORLUTIN™ (norethindrone), and NORLUTATE™ and AYGESTIN™ (norethindrone acetate); progesterone implants such as NORPLANT SYSTEM™ (subdermal implants of norgestrel); antiprogestins such as RU⁴⁸⁶™ (mifepristone); hormonal contraceptives such as ENOVID™ (norethynodrel plus mestranol), PROGESTASERT™ (intrauterine device that releases progesterone), LOESTRIN™, BREVICON™, MODICON™, GENORA™, NELONA™, NORINYL™, OVACON-35™ and OVACON-50™ (ethinyl estradiol/norethindrone), LEVLEN™, NORDETTE™, TRI-LEVLEN™ and TRIPHASIL-21™ (ethinyl estradiol/levonorgestrel) LO/OVRAL™ and OVRAL™ (ethinyl estradiol/norgestrel), DEMULEN™ (ethinyl estradiol/ethynodiol diacetate), NORINYL™, ORTHO-NOVUM™, NORETHIN™, GENORA™, and NELOVA™ (norethindrone/mestranol), DESOGEN™ and ORTHO-CEPT™ (ethinyl estradiol/desogestrel), ORTHO-CYCLEN™ and ORTHO-TRICYCLEN™ (ethinyl estradiol/norgestimate), MICRONOR™ and NOR-QD™ (norethindrone), and OVRETTE™ (norgestrel).

[1410] Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, testosterone esters such as methenolone acetate and testosterone undecanoate; parenteral and oral androgens such as TESTOJECT-50™ (testosterone), TESTEX™ (testosterone propionate), DELATESTRYL™ (testosterone enanthate), DEPO-TESTOSTERONE™ (testosterone cypionate), DANOCRINE™ (danazol), HALOTESTIN™ (fluoxymesterone), ORETON METHYL™, TESTRED™ and VIRILON™ (methyltestosterone), and OXANDRIN™ (oxandrolone); testosterone transdermal systems such as TESTODERM™; androgen receptor antagonist and 5-alpha-reductase inhibitors such as ANDROCUR™ (cyproterone acetate), EULEXIN™ (flutamide), and PROSCAR™ (finasteride); adrenocorticotropic hormone preparations such as CORTROSYN™ (cosyntropin); adrenocortical steroids and their synthetic analogs such as ACLOVATE™ (alclometasone dipropionate), CYCLOCORT™ (amcinonide), BECLOVENT™ and VANCERIL™ (beclomethasone dipropionate), CELESTONE™ (betamethasone), BENISONE™ and UTICORT™ (betamethasone benzoate), DIPROSONE™ (betamethasone dipropionate), CELESTONE PHOSPHATE™ (betamethasone sodium phosphate), CELESTONE SOLUSPAN™ (betamethasone sodium phosphate and acetate), BETA-VAL™ and VALISONE™ (betamethasone valerate), TEMOVATE™ (clobetasol propionate), CLODERM™0 (clocortolone pivalate), CORTEF™ and HYDROCORTONE™ (cortisol (hydrocortisone)), HYDROCORTONE ACETATE™ (cortisol (hydrocortisone) acetate), LOCOID™ (cortisol (hydrocortisone) butyrate), HYDROCORTONE PHOSPHATE™ (cortisol (hydrocortisone) sodium phosphate), A-HYDROCORT™ and SOLU CORTEF™ (cortisol (hydrocortisone) sodium succinate), WESTCORT™ (cortisol (hydrocortisone) valerate), CORTISONE ACETATE™ (cortisone acetate), DESOWEN™ and TRIDESILON™ (desonide), TOPICORT™ (desoximetasone), DECADRON™ (dexamethasone), DECADRON LA™ (dexamethasone acetate), DECADRON PHOSPHATE™ and HEXADROL PHOSPHATE™ (dexamethasone sodium phosphate), FLORONE™ and MAXIFLOR™ (diflorasone diacetate), FLORINEF ACETATE™ (fludrocortisone acetate), AEROBID™ and NASALIDE™ (flunisolide), FLUONID™ and SYNALAR™ (fluocinolone acetonide), LIDEX™ (fluocinonide), FLUOR-OP™ and FML™ (fluorometholone), CORDRAN™ (flurandrenolide), HALOG™ (halcinonide), HMS LIZUIFILM™ (medrysone), MEDROL™ (methylprednisolone), DEPO-MEDROL™ and MEDROL ACETATE™ (methylprednisone acetate), A-METHAPRED™ and SOLUMEDROL™ (methylprednisolone sodium succinate), ELOCON™ (mometasone furoate), HALDRONE™ (paramethasone acetate), DELTA-CORTEF™ (prednisolone), ECONOPRED™ (prednisolone acetate), HYDELTRASOL™ (prednisolone sodium phosphate), HYDELTRA-T.B.A™ (prednisolone tebutate), DELTASONE™ (prednisone), ARISTOCORT™ and KENACORT™ (triamcinolone), KENALOG™ (triamcinolone acetonide), ARISTOCORT™ and KENACORT DIACETATE™ (triamcinolone diacetate), and ARISTOSPAN™ (triamcinolone hexacetonide); inhibitors of biosynthesis and action of adrenocortical steroids such as CYTADREN™ (aminoglutethimide), NIZORAL™ (ketoconazole), MODRASTANE™ (trilostane), and METOPIRONE™ (metyrapone).

[1411] Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to bovine, porcine or human insulin or mixtures thereof; insulin analogs; recombinant human insulin such as HUMULIN™ and NOVOLIN™; oral hypoglycemic agents such as ORAMIDE™ and ORINASE™ (tolbutamide), DIABINESE™ (chlorpropamide), TOLAMIDE™ and TOLINASE™ (tolazamide), DYMELOR™ (acetohexamide), glibenclamide, MICRONASE™, DIBETA™ and GLYNASE™ (glyburide), GLUCOTROL™ (glipizide), and DIAMICRON™ (gliclazide), GLUCOPHAGE™ (metformin), PRECOSE™ (acarbose), AMARYL™ (glimepiride), and ciglitazone; thiazolidinediones (TZDs) such as rosiglitazone, AVANDIA™ (rosiglitazone maleate) ACTOS™ (piogliatazone), and troglitazone; alpha-glucosidase inhibitors; bovine or porcine glucagon; somatostatins such as SANDOSTATIN™ (octreotide); and diazoxides such as PROGLYCEM™ (diazoxide). In still other embodiments, Therapeutics of the invention are administered in combination with one or more of the following: a biguanide antidiabetic agent, a glitazone antidiabetic agent, and a sulfonylurea antidiabetic agent.

[1412] In one embodiment, the Therapeutics of the invention are administered in combination with treatments for uterine motility disorders. Treatments for uterine motility disorders include, but are not limited to, estrogen drugs such as conjugated estrogens (e.g., PREMARIN® and ESTRATAB™), estradiols (e.g., CLIMARA® and ALORA®), estropipate, and chlorotrianisene; progestin drugs (e.g., AMEN® (medroxyprogesterone), MICRONOR® (norethidrone acetate), PROMETRIUM® progesterone, and megestrol acetate); and estrogen/progesterone combination therapies such as, for example, conjugated estrogens/medroxyprogesterone (e.g., PREMPRO™ and PREMPHASE®) and norethindrone acetate/ethinyl estsradiol (e.g., FEMHRT™).

[1413] In an additional embodiment, the Therapeutics of the invention are administered in combination with drugs effective in treating iron deficiency and hypochromic anemias, including but riot limited to, ferrous sulfate (iron sulfate, FEOSOL™), ferrous fumarate (e.g., FEOSTAT™), ferrous gluconate (e.g., FERGON™), polysaccharide-iron complex (e.g., NIFEREX™), iron dextran injection (e.g., INFED™), cupric sulfate, pyroxidine, riboflavin, Vitamin B₁₂, cyancobalamin injection (e.g., REDISOL™, RUBRAMIN PC™), hydroxocobalamin, folic acid (e.g., FOLVITE™), leucovorin (folinic acid, 5-CHOH4PteGlu, citrovorum factor) or WELLCOVORIN (Calcium salt of leucovorin), transferrin or ferritin.

[1414] In certain embodiments, the Therapeutics of the invention are administered in combination with agents used to treat psychiatric disorders. Psychiatric drugs that may be administered with the Therapeutics of the invention include, but are not limited to, antipsychotic agents (e.g., chlorpromazine, chlorprothixene, clozapine, fluphenazine, haloperidol, loxapine, mesoridazine, molindone, olanzapine, perphenazine, pimozide, quetiapine, risperidone, thioridazine, thiothixene, trifluoperazine, and triflupromazine), antimanic agents (e.g., carbamazepine, divalproex sodium, lithium carbonate, and lithium citrate), antidepressants (e.g., amitriptyline, amoxapine, bupropion, citalopram, clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine, imipramine, isocarboxazid, maprotiline, mirtazapine, nefazodone, nortriptyline, paroxetine, phenelzine, protriptyline, sertraline, tranylcypromine, trazodone, trimipramine, and venlafaxine), antianxiety agents (e.g., alprazolam, buspirone, chlordiazepoxide, clorazepate, diazepam, halazepam, lorazepam, oxazepam, and prazepam), and stimulants (e.g., d-amphetamine, methylphenidate, and pemoline).

[1415] In other embodiments, the Therapeutics of the invention are administered in combination with agents used to treat neurological disorders. Neurological agents that may be administered with the Therapeutics of the invention include, but are not limited to, antiepileptic agents (e.g., carbamazepine, clonazepam, ethosuximide, phenobarbital, phenytoin, primidone, valproic acid, divalproex sodium, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide, diazepam, lorazepam, and clonazepam), antiparkinsonian agents (e.g., levodopa/carbidopa, selegiline, amantidine, bromocriptine, pergolide, ropinirole, pramipexole, benztropine; biperiden; ethopropazine; procyclidine; trihexyphenidyl, tolcapone), and ALS therapeutics (e.g. riluzole).

[1416] In another embodiment, Therapeutics of the invention are administered in combination with vasodilating agents and/or calcium channel blocking agents. Vasodilating agents that may be administered with the Therapeutics of the invention include, but are not limited to, Angiotensin Converting Enzyme (ACE) inhibitors (e.g., papaverine, isoxsuprine, benazepril, captopril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril, trandolapril, and nylidrin), and nitrates (e.g., isosorbide dinitrate, isosorbide mononitrate, and nitroglycerin). Examples of calcium channel blocking agents that may be administered in combination with the Therapeutics of the invention include, but are not limited to amlodipine, bepridil, diltiazem, felodipine, flunarizine, isradipine, nicardipine, nifedipine, nimodipine, and verapamil.

[1417] In additional embodiments, the Therapeutics of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

Example 24

[1418] Method of Treating Decreased Levels of the Polypeptide

[1419] The present invention relates to a method for treating an individual in need of an increased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an agonist of the invention (including polypeptides of the invention). Moreover, it will be appreciated that conditions caused by a decrease in the standard or normal expression level of a secreted protein in an individual can be treated by administering the polypeptide of the present invention, preferably in the secreted form. Thus, the invention also provides a method of treatment of an individual in need of an increased level of the polypeptide comprising administering to such an individual a Therapeutic comprising an amount of the polypeptide to increase the activity level of the polypeptide in such an individual.

[1420] For example, a patient with decreased levels of a polypeptide receives a daily dose 0.1-100 ug/kg of the polypeptide for six consecutive days. Preferably, the polypeptide is in the secreted form. The exact details of the dosing scheme, based on administration and formulation, are provided in Example 23.

Example 25

[1421] Method of Treating Increased Levels of the Polypeptide

[1422] The present invention also relates to a method of treating an individual in need of a decreased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an antagonist of the invention (including polypeptides and antibodies of the invention).

[1423] In one, example, antisense technology is used to inhibit production of a polypeptide of the present invention. This technology is one example of a method of decreasing levels of a polypeptide, preferably a secreted form, due to a variety of etiologies, such as cancer. For example, a patient diagnosed with abnormally increased levels of a polypeptide is administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the treatment was well tolerated. The formulation of the antisense polynucleotide is provided in Example 23.

Example 26

[1424] Method of Treatment Using Gene Therapy-Ex Vivo

[1425] One method of gene therapy transplants fibroblasts, which are capable of expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37 degree C. for approximately one week.

[1426] At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.

[1427] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and Hindi HI and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.

[1428] The cDNA encoding a polypeptide of the present invention can be amplified using PCR primers which correspond to the 5′ and 3′ end sequences respectively as set forth in Example 1 using primers and having appropriate restriction sites and initiation/stop codons, if necessary. Preferably, the 5′ primer contains an EcoRI site and the 3′ primer includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted.

[1429] The amphotropic pA317 or GP+am12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).

[1430] Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether protein is produced.

[1431] The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.

Example 27

[1432] Gene Therapy Using Endogenous Genes Corresponding To Polynucleotides of the Invention

[1433] Another method of gene therapy according to the present invention involves operably associating the endogenous polynucleotide sequence of the invention with a promoter via homologous recombination as described, for example, in U.S. Pat. NO: 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/2941 1, published Sep. 26, 1996; International Publication NO: WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not expressed in the cells, or is expressed -at a lower level than desired.

[1434] Polynucleotide constructs are made which contain a promoter and targeting sequences, which are homologous to the 5′ non-coding sequence of endogenous polynucleotide sequence, flanking the promoter. The targeting sequence will be sufficiently near the 5′ end of the polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination. The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter.

[1435] The amplified promoter and the amplified targeting sequences are digested with the appropriate restriction enzymes and subsequently treated with calf intestinal phosphatase. The digested promoter and digested targeting sequences are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The construct is size fractionated on an agarose gel then purified by phenol extraction and ethanol precipitation.

[1436] In this Example, the polynucleotide constructs are administered as naked polynucleotides via electroporation. However, the polynucleotide constructs may also be administered with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, precipitating agents, etc. Such methods of delivery are known in the art.

[1437] Once the cells are transfected, homologous recombination will take place which results in the promoter being operably linked to the endogenous polynucleotide sequence. This results in the expression of polynucleotide corresponding to the polynucleotide in the cell. Expression may be detected by immunological staining, or any other method known in the art.

[1438] Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in DMEM+10% fetal calf serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the plastic surface with nutrient medium. An aliquot of the cell suspension is removed for counting, and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg/ml acetylated bovine serum albumin. The final cell suspension contains approximately 3×10⁶ cells/ml. Electroporation should be performed immediately following resuspension.

[1439] Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the locus corresponding to the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst, NY) is digested with HindIII. The CMV promoter is amplified by PCR with an XbaI site on the 5′ end and a BamHI site on the 3′end. Two non-coding sequences are amplified via PCR: one non-coding sequence (fragment 1) is amplified with a HindIII site at the 5′ end and an Xba site at the 3′end; the other non-coding sequence (fragment 2) is amplified with a BamHI site at the 5′end and a HindIII site at the 3′end. The CMV promoter and the fragments (1 and 2) are digested with the appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI; fragment 2—BamHI) and ligated together. The resulting ligation product is digested with HindIII, and ligated with the HindIII-digested pUC18 plasmid.

[1440] Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-Rad). The final DNA concentration is generally at least 120 μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5.×10⁶ cells) is then added to the cuvette, and the cell suspension and DNA solutions are gently mixed. Electroporation is performed with a Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and 250-300 V, respectively. As voltage increases, cell survival decreases, but the percentage of surviving cells that stably incorporate the introduced DNA into their genome increases dramatically. Given these parameters, a pulse time of approximately 14-20 mSec should be observed.

[1441] Electroporated cells are maintained at room temperature for approximately 5 min, and the contents of the cuvette are then gently removed with a sterile transfer pipette. The cells are added directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37 degree C. The following day, the media is aspirated and replaced with 10 ml of fresh media and incubated for a further 16-24 hours.

[1442] The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product. The fibroblasts can then be introduced into a patient as described above.

Example 28

[1443] Method of Treatment Using Gene Therapy-In Vivo

[1444] Another aspect of the present invention is using in vivo gene therapy methods to treat disorders, diseases and conditions. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease the expression of the polypeptide. The polynucleotide of the present invention may be operatively linked to a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. No. 5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res. 35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., Gene Ther. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290 (1996) (incorporated herein by reference).

[1445] The polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

[1446] The term “naked” polynucleotide, DNA or RNA, refers to sequences that are 10 free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the present invention may also be delivered in liposome formulations (such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods well known to those skilled in the art.

[1447] The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapies techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

[1448] The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.

[1449] For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.

[1450] The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for production of mRNA coding for polypeptide -of the present invention is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA.

[1451] Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips.

[1452] After an appropriate incubation time (e.g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 um cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course for protein expression may be done in a similar fashion except that quadriceps from different mice are harvested at different times. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experimentation in mice can be use to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA.

Example 29

[1453] Transgenic Animals.

[1454] The polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.

[1455] Any technique known in the art may be used to introduce the transgene (i.e., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by reference herein in its entirety.

[1456] Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

[1457] The present invention provides for transgenic animals that carry the transgene i n all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

[1458] Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

[1459] Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.

[1460] Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying diseases, disorders, and/or conditions associated with aberrant expression, and in screening for compounds effective in ameliorating such diseases, disorders, and/or conditions.

Example 30

[1461] Knock-Out Animals.

[1462] Endogenous gene expression can also be reduced by inactivating or “knocking out” the gene and/or its promoter using targeted homologous recombination. (E.g., see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art.

[1463] In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.

[1464] Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated by reference herein in its entirety).

[1465] When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.

[1466] Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying diseases, disorders, and/or conditions associated with aberrant expression, and in screening for compounds effective in ameliorating such diseases, disorders, and/or conditions.

Example 31

[1467] Production of an Antibody

[1468] Hybridoma Technology

[1469] The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, cells expressing polypeptide(s) of the invention are administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of polypeptide(s) of the invention is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.

[1470] Monoclonal antibodies specific for polypeptide(s) of the invention are prepared using hybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is immunized with polypeptide(s) of the invention, or, more preferably, with a secreted polypeptide-expressing cell. Such polypeptide-expressing cells are cultured in any suitable tissue culture medium, preferably in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56° C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.

[1471] The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 8.0:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the polypeptide(s) of the invention.

[1472] Alternatively, additional antibodies capable of binding polypeptide(s) of the invention can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the polypeptide(s) of the invention protein-specific antibody can be blocked by polypeptide(s) of the invention. Such antibodies comprise anti-idiotypic antibodies to the polypeptide(s) of the invention protein-specific antibody and are used to immunize an animal to induce formation of further polypeptide(s) of the invention protein-specific antibodies.

[1473] For in vivo use of antibodies in humans, an antibody is “humanized”. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).)

[1474] Isolation Of Antibody Fragments Directed polypeptide(s) of the invention From A Library Of scFvs

[1475] Naturally occurring V-genes isolated from human PBLs are constructed into a library of antibody fragments which contain reactivities against polypeptide(s) of the invention to which the donor may or may not have been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein by reference in its entirety).

[1476] Rescue of the Library. A library of scFvs is constructed from the RNA of human PBLs as described in PCT publication WO 92/01047. To rescue phage displaying antibody fragments, approximately 109 E. coli harboring the phagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and 100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of this culture is used to innoculate 50 ml of 2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, see PCT publication WO 92/01047) are added and the culture incubated at 37° C. for 45 minutes without shaking and then at 37° C. for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are prepared as described in PCT publication WO 92/01047.

[1477] M13 delta gene III is prepared as follows: M13 delta gene III helper phage does not encode gene III protein, hence the phage(mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious M13 delta gene III particles are made by growing the helper phage in cells harboring a pUC19 derivative supplying the wild type gene III protein during phage morphogenesis. The culture is incubated for 1 hour at 37° C. without shaking and then for a further hour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C. Phage particles are purified and concentrated from -the culture medium by two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS and passed through a 0.45 μm filter (Minisart NML; Sartorius) to give a final concentration of approximately 1013 transducing units/ml (ampicillin-resistant clones).

[1478] Panning of the Library. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of the present invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage is applied to the tube and incubated for 30 minutes at room temperature tumbling on an over and under turntable and then left to stand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1 by incubating eluted phage with bacteria for 30 minutes at 37° C. The E. coli are then plated on TYE plates containing 1% glucose and 100 μg/ml ampicillin. The resulting bacterial library is then rescued with delta gene 3 helper phage as described above to prepare phage for a subsequent round of selection. This process is then repeated for a total of 4 rounds of affinity purification with tube-washing increased to 20 times with PBS, 0. 1% Tween-20 and 20 times with PBS for rounds 3 and 4.

[1479] Characterization of Binders. Eluted phage from the 3rd and 4th rounds of selection are used to infect E. coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single colonies for assay. ELISAs are performed with microtitre plates coated with either 10 pg/ml of the polypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISA are further characterized by PCR fingerprinting (see, e.g., PCT publication WO 92/01047) and then by sequencing. These ELISA positive clones may also be further characterized by techniques known in the art, such as, for example, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody/antigen binding, and competitive agonistic or antagonistic activity.

Example 32

[1480] Assays Detecting Stimulation or Inhibition of B cell Proliferation and Differentiation

[1481] Generation of functional humoral immune responses requires both soluble and cognate signaling between B-lineage cells and their microenvironment. Signals may impart a positive stimulus that allows a B-lineage cell to continue its programmed development, or a negative stimulus that instructs the cell to arrest its current developmental pathway. To date, numerous stimulatory and inhibitory signals have been found to influence B cell responsiveness including IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signals are by themselves weak effectors but can, in combination with various co-stimulatory proteins, induce activation, proliferation, differentiation, homing, tolerance and death among B cell populations.

[1482] One of the best studied classes of B-cell co-stimulatory proteins is the TNF-superfamily. Within this family CD40, CD27, and CD30 along with their respective ligands CD154, CD70, and CD153 have been found to regulate a variety of immune responses. Assays which allow for the detection and/or observation of the proliferation and differentiation of these B-cell populations and their precursors are valuable tools in determining the effects various proteins may have on these B-cell populations in terms of proliferation and differentiation. Listed below are two assays designed to allow for the detection of the differentiation, proliferation, or inhibition of B-cell populations and their precursors.

[1483] In Vitro Assay-Purified polypeptides of the invention, or truncated forms thereof, is assessed for its ability to induce activation, proliferation, differentiation or inhibition and/or death in B-cell populations and their precursors. The activity of the polypeptides of the invention on purified human tonsillar B cells, measured qualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay in which purified tonsillar B cells are cultured in the presence of either formalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilized anti-human IgM antibody as the priming agent. Second signals such as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cell proliferation as measured by tritiated-thymidine incorporation. Novel synergizing agents can be readily identified using this assay. The assay involves isolating human tonsillar B cells by magnetic bead (MACS) depletion of CD3-positive cells. The resulting cell population is greater than 95% B cells as assessed by expression of CD45R(B220).

[1484] Various dilutions of each sample are placed into individual wells of a 96-well plate to which are added 10⁵ B-cells suspended in culture medium (RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100 U/ml penicillin, 10 ug/ml streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of 150 ul. Proliferation or inhibition is quantitated by a 20 h pulse (1 uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factor addition. The positive and negative controls are IL2 and medium respectively.

[1485] In Vivo Assay-BALB/c mice are injected (i.p.) twice per day with buffer only, or 2 mg/Kg of a polypeptide of the invention, or truncated forms thereof. Mice receive this treatment for 4 consecutive days, at which time they are sacrificed and various tissues and serum collected for analyses. Comparison of H&E sections from normal spleens and spleens treated with polypeptides of the invention identify the results of the activity of the polypeptides on spleen cells, such as the diffusion of peri-arterial lymphatic sheaths, and/or significant increases in the nucleated cellularity of the red pulp regions, which may indicate the activation of the differentiation and proliferation of B-cell populations. Immunohistochemical studies using a B cell marker, anti-CD45R(B220), are used to determine whether any physiological changes to splenic cells, such as splenic disorganization, are due to increased B-cell representation within loosely defined B-cell zones that infiltrate established T-cell regions.

[1486] Flow cytometric analyses of the spleens from mice treated with polypeptide is used to indicate whether the polypeptide specifically increases the proportion of ThB+, CD45R(B220)dull B cells over that which is observed in control mice.

[1487] Likewise, a predicted consequence of increased mature B-cell representation in vivo is a relative increase in serum Ig titers. Accordingly, serum IgM and IgA levels are compared between buffer and polypeptide-treated mice.

[1488] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 33

[1489] T Cell Proliferation Assay

[1490] Proliferation assay for Resting PBLs.

[1491] A CD3-induced proliferation assay is performed on PBMCs and is measured by the uptake of ³H-thymidine. The assay is performed as follows. Ninety-six well plates are coated with 100 microliters per well of mAb to CD3 (HIT₃a, Pharmingen) or isotype-matched control mAb (B33. 1) overnight at 4° C. (1 microgram/ml in 0.05M bicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC are isolated by F/H gradient centrifugation from human peripheral blood and added to quadruplicate wells (5×10⁴/well) of mAb coated plates in RPMI containing 10% FCS and P/S in the presence of varying concentrations of TNF Delta and/or TNF Epsilon protein (total volume 200 microliters). Relevant protein buffer and medium alone are controls. After 48 hr. culture at 37° C., plates are spun for 2 min. at 1000 rpm and 100 microliters of supernatant is removed and stored ˜20° C. for measurement of IL-2 (or other cytokines) if effect on proliferation is observed. Wells are supplemented with 100 microliters of medium containing 0.5 microcuries of ³H-thymidine and cultured at 37° C. for 18-24 hr. Wells are harvested and incorporation of ³H-thymidine used as a measure of proliferation. Anti-CD3 alone is the positive control for proliferation. IL-2 (100 U/ml) is also used as a control which enhances proliferation. Control antibody which does not induce proliferation of T cells is used as the negative controls for the effects of TNF Delta and/or TNF Epsilon proteins.

[1492] Alternatively, a proliferation assay on resting PBL (peripheral blood lymphocytes) is measured by the up-take of ³H-thymidine. The assay is performed as follows. PBMC are isolated by Ficoll (LSM, ICN Biotechnologies, Aurora, Ohio) gradient centrifugation from human peripheral blood, and are cultured overnight in 10% (Fetal Calf Serum, Biofluids, Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg, Md.). This overnight incubation period allows the adherent cells to attach to the plastic, which results in a lower background in the assay as there are fewer cells that can act as antigen presenting cells or that might be producing growth factors. The following day the non-adherent cells are collected, washed and used in the proliferation assay. The assay is performed in a 96 well plate using 2×10⁴ cells/well in a final volume of 200 microliters. The supernatants (e.g., CHO or 293T supernatants) expressing the protein of interest are tested at a 30% final dilution, therefore 60 ul are added to 140 ul of 10% FCS/RPMI containing the cells , Control supernatants are used at the same final dilution and express the following proteins: vector (negative control), IL-2 (*), IFN□, TNF□, IL-10 and TR2. In addition to the control supernatants, recombinant human IL-2 (R & D Systems, Minneapolois, Minn.) at a final concentration of 100 ng/ml is also used. After 24 hours of culture, each well is pulsed with 1 uCi of ³H-thymidine (Nen, Boston, Mass.). Cells are then harvested 20 hours following pulsing and incorporation of ³H-thymidine is used as a measure of proliferation. Results are expressed as an average of triplicate samples plus or minus standard error.

[1493] (*) The amount of the control cytokines IL-2, IFN□, TNF□ and IL-10 produced in each transfection varies between 300 pg to 5 ng/ml.

[1494] Costimulation Assay.

[1495] A costimulation assay on resting PBL (peripheral blood lymphocytes) is performed in the presence of immobilized antibodies to CD3 and CD28. The use of antibodies specific for the invariant regions of CD3 mimic the induction of T cell activation that would occur through stimulation of the T cell receptor by an antigen. Cross-linking of the TCR (first signal) in the absence of a costimulatory signal (second signal) causes very low induction of proliferation and will eventually result in a state of “anergy”, which is characterized by the absence of growth and inability to produce cytokines. The addition of a costimulatory signal such as an antibody to CD28, which mimics the action of the costimulatory molecule. B7-1 expressed on activated APCs, results in enhancement of T cell responses including cell survival and production of IL-2. Therefore this type of assay allows to detect both positive and negative effects caused by addition of supernatants expressing the proteins of interest on T cell proliferation.

[1496] The assay is performed as follows. Ninety-six well plates are coated with 100 ng/ml anti-CD3 and 5 ug/ml anti-CD28 (Pharmingen, San Diego, Calif.) in a final volume of 100 ul and incubated overnight at 4C. Plates are washed twice with PBS before use. PBMC are isolated by Ficoll (LSM, ICN Biotechnologies, Aurora, Ohio) gradient centrifugation from human peripheral blood, and are cultured overnight in 10% FCS(Fetal Calf Serum, Biofluids, Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg, Md.). This overnight incubation period allows the adherent cells to attach to the plastic, which results in a lower background in the assay as there are fewer cells that can act as antigen presenting cells or that might be producing growth factors. The following day the non adherent cells are collected, washed and used in the proliferation assay. The assay is performed in a 96 well plate using 2×10⁴ cells/well in a final volume of 200 ul. The supernatants (e.g., CHO or 293T supernatants) expressing the protein of interest are tested at a 30% final dilution, therefore 60 ul are added to 140 ul of 10% FCS/RPMI containing the cells. Control supernatants are used at the same final dilution and express the following proteins: vector only (negative control), IL-2, TFN□, TNF□, IL-10 and TR2. In addition to the control supernatants recombinant human IL-2 (R & D Systems, Minneapolis, Minn.) at a final concentration of 10 ng/ml is also used. After 24 hours of culture, each well is pulsed with 1 uCi of ³H-thymidine (Nen, Boston, Mass.). Cells are then harvested 20 hours following pulsing and incorporation of ³H-thymidine is used as a measure of proliferation. Results are expressed as an average of triplicate samples plus or minus standard error.

[1497] Costimulation assay: IFN γ and IL-2 ELISA.

[1498] The assay is performed as follows. Twenty-four well plates are coated with either 300 ng/ml or 600 ng/ml anti-CD3 and 5 ug/ml anti-CD28 (Pharmingen, San Diego, Calif.) in a final volume of 500 ul and incubated overnight at 4C. Plates are washed twice with PBS before use. PBMC are isolated by Ficoll (LSM, ICN Biotechnologies, Aurora, Ohio) gradient centrifugation from human peripheral blood, and are cultured overnight in 10% FCS(Fetal Calf Serum, Blofluids, Rockville. Md.)/RPMI (Gibco BRL, Gaithersburg, Md.). This overnight incubation period allows the adherent cells to attach to the plastic, which results in a lower background in the assay as there are fewer cells that can act as antigen presenting cells or that might be producing growth factors. The following day the non adherent cells are collected, washed and used in the costimulation assay. The assay is performed in the pre-coated twenty-four well plate using 1×10⁵ cells/well in a final volume of 900 ul. The supernatants (293T supernatants) expressing the protein of interest are tested at a 30% final dilution, therefore 300 ul are added to 600 ul of 10% FCS/RPMI containing the cells. Control supernatants are used at the same final dilution and express the following proteins: vector only(negative control), IL-2, IFN□, IL-12 and IL-18. In addition to the control supernatants recombinant human IL-2 (all cytokines were purchased from R & D Systems, Minneapolis, Minn.) at a final concentration of 10 ng/ml, IL-12 at a final concentration of 1 ng/ml and IL-18 at a final concentration of 50 ng/ml are also used. Controls and unknown samples are tested in duplicate. Supernatant samples (250 ul) are collected 2 days and 5 days after the beginning of the assay. ELISAs to test for IFN□ and IL-2 secretion are performed using kits purchased from R & D Systems, (Minneapolis, Minn.). Results are expressed as an average of duplicate samples plus or minus standard error.

[1499] Proliferation Assay for Preactivated-Resting T cells.

[1500] A proliferation assay on preactivated-resting T cells is performed on cells that are previously activated with the lectin phytohemagglutinin (PHA). Lectins are polymeric plant proteins that can bind to residues on T cell surface glycoproteins including the TCR and act as polyclonal activators. PBLs treated with PHA and then cultured in the presence of low doses of IL-2 resemble effector T cells. These cells are generally more sensitive to further activation induced by growth factors such as IL-2. This is due to the expression of high affinity IL-2 receptors that allows this population to respond to amounts of IL-2 that are 100 fold lower than what would have an effect on a naïve T cell. Therefore the use of this type of cells might enable to detect the effect of very low doses of an unknown growth factor, that would not be sufficient to induce proliferation on resting (naïve) T cells.

[1501] The assay is performed as follows. PBMC are isolated by F/H gradient centrifugation from human peripheral blood, and are cultured in 10% FCS(Fetal Calf Serum, Biofluids, Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg, Md.) in the presence of 2 ug/ml PHA (Sigma, Saint Louis, Mo.) for three days. The cells are then washed in PBS and cultured in 10% FCS/RPMI in the presence of 5 ng/ml of human recombinant IL-2 (R & D Systems, Minneapolis, Minn.) for 3 days. The cells are washed and rested in starvation medium (1%FCS/RPMI) for 16 hours prior to the beginning of the proliferation assay. An aliquot of the cells is analyzed by FACS to determine the percentage of T cells (CD3 positive cells) present; this usually ranges between 93-97% depending on the donor. The assay is performed in a 96 well plate using 2×10⁴ cells/well in a final volume of 200 ul. The supernatants (e.g., CHO or 293T supernatants) expressing the protein of interest are tested at a 30% final dilution, therefore 60 ul are added to 140 ul of in 10% FCS/RPMI containing the cells. Control supernatants are used at the same final dilution and express the following proteins: vector (negative control), IL-2, IFN□, TNF□, IL-10 and TR2. In addition to the control supernatants recombinant human IL-2 at a final concentration of 10 ng/ml is also used. After 24 hours of culture, each well is pulsed with 1 uCi of ³H-thymidine(Nen, Boston, Mass.). Cells are then harvested 20 hours following pulsing and incorporation of ³H-thymidine is used as a measure of proliferation. Results are expressed as an average of triplicate samples plus or minus standard error.

[1502] The studies described in this example test activity of polypeptides of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 34

[1503] Effect of Polypeptides of the Invention on the Expression of MHC Class II, Costimulatory and Adhesion Molecules and Cell Differentiation of Monocytes and Monocyte-Derived Human Dendritic Cells

[1504] Dendritic cells are generated by the expansion of proliferating precursors found in the peripheral blood: adherent PBMC or elutriated monocytic fractions are cultured for 7-10 days with GM-CS F (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells have the characteristic phenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with activating factors, such as TNF-α, causes a rapid change in surface phenotype (increased expression of MHC class I and II, costimulatory and adhesion molecules, downregulation of FCγRII, upregulation of CD83). These changes correlate with increased antigen-presenting capacity and with functional maturation of the dendritic cells.

[1505] FACS analysis of surface antigens is performed as follows. Cells are treated 1-3 days with increasing concentrations of polypeptides of the invention or LPS (positive control), washed with PBS containing 1 % BSA and 0.02 miM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

[1506] Effect on the production of cytokines. Cytokines generated by dendritic cells, in particular IL-12, are important in the initiation of T-cell dependent immune responses. IL-12 strongly influences the development of Th1 helper T-cell immune response, and induces cytotoxic T and NK cell function. An ELISA is used to measure the IL-12 release as follows. Dendritic cells (10⁶/ml) are treated with increasing concentrations of polypeptides of the invention for 24 hours. LPS (100 ng/ml) is added to the cell culture as positive control. Supernatants from the cell cultures are then collected and analyzed for IL-12 content using commercial ELISA kit (e..g, R & D Systems (Minneapolis, Minn.)). The standard protocols provided with the kits are used.

[1507] Effect on the expression of MHC Class II, costimulatory and adhesion molecules. Three major families of cell surface antigens can be identified on monocytes: adhesion molecules, molecules involved in antigen presentation, and Fc receptor. Modulation of the expression of MHC class II antigens and other costimulatory molecules, such as B7 and ICAM-1, may result in changes in the antigen presenting capacity of monocytes and ability to induce T cell activation. Increase expression of Fc receptors may correlate with improved monocyte cytotoxic activity, cytokine release and phagocytosis.

[1508] FACS analysis is used to examine the surface antigens as follows. Monocytes are treated 1-5 days with increasing concentrations of polypeptides of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

[1509] Monocyte activation and/or increased survival. Assays for molecules that activate (or alternatively, inactivate) monocytes and/or increase monocyte survival (or alternatively, decrease monocyte survival) are known in the art and may routinely be applied to determine whether a molecule of the invention functions as an inhibitor or activator of monocytes. Polypeptides, agonists, or antagonists of the invention can be screened using the three assays described below. For each of these assays, Peripheral blood mononuclear cells (PBMC) are purified from single donor leukopacks (American Red Cross, Baltimore, Md.) by centrifugation through a Histopaque gradient (Sigma). Monocytes are isolated from PBMC by counterflow centrifugal elutriation.

[1510] Monocyte Survival Assay. Human peripheral blood monocytes progressively lose viability when cultured in absence of serum or other stimuli. Their death results from internally regulated process (apoptosis). Addition to the culture of activating factors, such as TNF-alpha dramatically improves cell survival and prevents DNA fragmentation. Propidium iodide (PI) staining is used to measure apoptosis as follows. Monocytes are cultured for 48 hours in polypropylene tubes in serum-free medium (positive control), in the presence of 100 ng/ml TNF-alpha (negative control), and in the presence of varying concentrations of the compound to be tested. Cells are suspended at a concentration of 2×10⁶/ml in PBS containing PI at a final concentration of 5 μg/ml, and then incubaed at room temperature for 5 minutes before FACScan analysis. PI uptake has been demonstrated to correlate with DNA fragmentation in this experimental paradigm.

[1511] Effect on cytokine release. An important function of monocytes/macrophages is their regulatory activity on other cellular populations of the immune system through the release of cytokines after stimulation. An ELISA to measure cytokine release is performed as follows. Human monocytes are incubated at a density of 5×10⁵ cells/ml with increasing concentrations of the a polypeptide of the invention and under the same conditions, but in the absence of the polypeptide. For IL-12 production, the cells are primed overnight with IFN (100 U/ml) in presence of a polypeptide of the invention. LPS (10 ng/ml) is then added. Conditioned media are collected after 24 h and kept frozen until use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a commercially available ELISA kit (e..g, R & D Systems (Minneapolis, Minn.)) and applying the standard protocols provided with the kit.

[1512] Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1×10⁵ cell/well. Increasing concentrations of polypeptides of the invention are added to the wells in a total volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamine and antibiotics). After 3 days incubation, the plates are centrifuged and the medium is removed from the wells. To the macrophage monolayers, 0.2 ml per well of phenol red solution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA). The plates are incubated at 37° C. for 2 hours and the reaction is stopped by adding 20 μl IN NaOH per well. The absorbance is read at 610 nm. To calculate the amount of H₂O₂ produced by the macrophages, a standard curve of a H₂O₂ solution of known molarity is performed for each experiment.

[1513] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polypeptides, polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 35

[1514] Biological Effects of Polypeptides of the Invention

[1515] Astrocyte and Neuronal Assays:

[1516] Recombinant polypeptides of the invention, expressed in Escherichia coli and purified as described above, can be tested for activity in promoting the survival, neurite outgrowth, or phenotypic differentiation of cortical neuronal cells and for inducing the proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes. The selection of cortical cells for the bioassay is based on the prevalent expression of FGF-1 and FGF-2 in cortical structures and on the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. A thymidine incorporation assay, for example, can be used to elucidate a polypeptide of the invention's activity on these cells.

[1517] Moreover, previous reports describing the biological effects of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro have demonstrated increases in both neuron survival and neurite outgrowth (Walicke et al., “Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension.” Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated by reference in its entirety). However, reports from experiments done on PC-12 cells suggest that these two responses are not necessarily synonymous and may depend on not only which FGF is being tested but also on which receptor(s) are expressed on the target cells. Using the primary cortical neuronal culture paradigm, the ability of a polypeptide of the invention to induce neurite outgrowth can be compared to the response achieved with FGF-2 using, for example, a thymidine incorporation assay.

[1518] Fibroblast and endothelial cell assays

[1519] Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.) and maintained in growth media from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, Calif.). For proliferation assays, the human lung fibroblasts and dermal -microvascular endothelial cells can be cultured at 5,000 cells/well in a 96-well plate for one day in growth medium. The cells are then incubated for one day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) is added to each well to a final concentration of 10%. The cells are incubated for 4 hr. Cell viability is measured by reading in a CytoFluor fluorescence reader. For the PGE₂ assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or polypeptides of the invention with or without IL-1α for 24 hours. The supernatants are collected and assayed for PGE₂ by EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or with or without polypeptides of the invention IL-1α for 24 hours. The supernatants are collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, Mass.).

[1520] Human lung fibroblasts are cultured with FGF-2 or polypeptides of the invention for 3 days in basal medium before the addition of Alamar Blue to assess effects on growth of the fibroblasts. FGF-2 should show a stimulation at 10-2500 ng/ml which can be used to compare stimulation with polypeptides of the invention.

[1521] Parkinson Models.

[1522] The loss of motor function in Parkinson's disease is attributed to a deficiency of striatal dopamine resulting from the degeneration of the nigrostriatal dopaminergic projection neurons. An animal model for Parkinson's that has been extensively characterized involves the systemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized by monoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP⁺) and released. Subsequently, MPP⁺ is actively accumulated in dopaminergic neurons by the high-affinity reuptake transporter for dopamine. MPP⁺ is then concentrated in mitochondria by the electrochemical gradient and selectively inhibits nicotidamide adenine disphosphate: ubiquinone oxidoreductionase (complex I), thereby interfering with electron transport and eventually generating oxygen radicals.

[1523] It has been demonstrated in tissue culture paradigms that FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group has demonstrated that administering FGF-2 in gel foam implants in the striatum results in the near complete protection of nigral dopaminergic neurons from the toxicity associated with MPTP exposure (Otto and Unsicker, J. Neuroscience, 1990).

[1524] Based on the data with FGF-2, polypeptides of the invention can be evaluated to determine whether it has an action similar to that of FGF-2 in enhancing dopaminergic neuronal survival in vitro and it can also be tested in vivo for protection of dopaminergic neurons in the striatum from the damage associated with MPTP treatment. The potential effect of a polypeptide of the invention is first examined in vitro in a dopaminergic neuronal cell culture paradigm. The cultures are prepared by dissecting the midbrain floor plate from gestation day 14 Wistar rat embryos. The tissue is dissociated with trypsin and seeded at a density of 200,000 cells/cm² on polyorthinine-laminin coated glass coverslips. The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium containing hormonal supplements (N1). The cultures are fixed with paraformaldehyde after 8 days in vitro and are processed for tyrosine hydroxylase, a specific marker for dopminergic neurons, immunohistochemical staining. Dissociated cell cultures are prepared from embryonic rats. The culture medium is changed every third day and the factors are also added at that time.

[1525] Since the dopaminergic neurons are isolated from animals at gestation day 14, a developmental time which is past the stage when the dopaminergic precursor cells are proliferating, an increase in the number of tyrosine hydroxylase immunopositive neurons would represent an increase in the number of dopaminergic neurons surviving in vitro. Therefore, if a polypeptide of the invention acts to prolong the survival of dopaminergic neurons, it would suggest that the polypeptide may be involved in Parkinson's Disease.

[1526] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 36

[1527] The Effect of Polypeptides of the Invention on the Growth of Vascular Endothelial Cells

[1528] On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at 2-5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium is replaced with M199 containing 10% FBS, 8 units/ml heparin. A polypeptide having the amino acid sequence of SEQ ID NO: Y, and positive controls, such as VEGF and basic FGF (bFGF) are added, at varying concentrations. On days 4 and 6, the medium is replaced. On day 8, cell number is determined with a Coulter Counter.

[1529] An increase in the number of HUVEC cells indicates that the polypeptide of the invention may proliferate vascular endothelial cells.

[1530] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 37

[1531] Stimulatory Effect of Polypeptides of the Invention on the Proliferation of Vascular Endothelial Cells

[1532] For evaluation of mitogenic activity of growth factors, the colorimetric MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) assay with the electron coupling reagent PMS (phenazine methosulfate) was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-well plate (5,000 cells/well) in 0.1 mL serum-supplemented medium and are allowed to attach overnight. After serum-starvation for 12 hours in 0.5% FBS, conditions (bFGF, VEGF₁₆₅ or a polypeptide of the invention in 0.5% FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMS mixture (1:0.05) are added per well and allowed to incubate for 1 hour at 37° C. before measuring the absorbance at 490 nm in an ELISA plate reader. Background absorbance from control wells (some media, no cells) is subtracted, and seven wells are performed in parallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol. 30A:512-518 (1994).

[1533] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 38

[1534] Inhibition of PDGF-Induced Vascular Smooth Muscle Cell Proliferation Stimulatory Effect

[1535] HAoSMC proliferation can be measured, for example, by BrdUrd incorporation. Briefly, subconfluent, quiescent cells grown on the 4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then, the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h, immunocytochemistry is performed by using BrdUrd Staining Kit (Zymed Laboratories). In brief, the cells are incubated with the biotinylated mouse anti-BrdUrd antibody at 4 degrees C. for 2 h after being exposed to denaturing solution and then incubated with the streptavidin-peroxidase and diaminobenzidine. After counterstaining with hematoxylin, the cells are mounted for microscopic examination, and the BrdUrd-positive cells are counted. The BrdUrd index is calculated as a percent of the BrdUrd-positive cells to the total cell number. In addition, the simultaneous detection of the BrdUrd staining (nucleus) and the FITC uptake (cytoplasm) is performed for individual cells by the concomitant use of bright field illumination and dark field-UV fluorescent illumination. See, Hayashida et al., J. Biol. Chem. 6:271(36):21985-21992 (1996).

[1536] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 39

[1537] Stimulation of Endothelial Migration

[1538] This example will be used to explore the possibility that a polypeptide of the invention may stimulate lymphatic endothelial cell migration.

[1539] Endothelial cell migration assays are performed using a 48 well microchemotaxis chamber (Neuroprobe Inc., Cabin John, M D; Falk, W., et al., J. Immunological Methods 1980;33:239-247). Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um (Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for at least 6 hours at room temperature and dried under sterile air. Test substances are diluted to appropriate concentrations in M199 supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of the final dilution is placed in the lower chamber of the modified Boyden apparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for the minimum time required to achieve cell detachment. After placing the filter between lower and upper chamber, 2.5×10⁵ cells suspended in 50 ul M199 containing 1% FBS are seeded in the upper compartment. The apparatus is then incubated for 5 hours at 37° C. in a humidified chamber with 5% CO2 to allow cell migration. After the incubation period, the filter is removed and the upper side of the filter with the non-migrated cells is scraped with a rubber policeman. The filters are fixed with methanol and stained with a Giemsa solution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration is quantified by counting cells of three random high-power fields (40×) in each well, and all groups are performed in quadruplicate.

[1540] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 40

[1541] Stimulation of Nitric Oxide Production by Endothelial Cells

[1542] Nitric oxide released by the vascular endothelium is believed to be a mediator of vascular endothelium relaxation. Thus, activity of a polypeptide of the invention can be assayed by determining nitric oxide production by endothelial cells in response to the polypeptide.

[1543] Nitric oxide is measured in 96-well plates of confluent microvascular endothelial cells after 24 hours starvation and a subsequent 4 hr exposure to various levels of a positive control (such as VEGF-1) and the polypeptide of the invention. Nitric oxide in the medium is determined by use of the Griess reagent to measure total nitrite after reduction of nitric oxide-derived nitrate by nitrate reductase. The effect of the polypeptide of the invention on nitric oxide release is examined on HUVEC.

[1544] Briefly, NO release from cultured HUVEC monolayer is measured with a NO-specific polarographic electrode connected to a NO meter (Iso-NO, World Precision Instruments Inc.) (1049). Calibration of the NO elements is performed according to the following equation:

2KNO₂+2KI+2H₂SO₄6 2NO+I₂+2H₂O+2K₂SO₄

[1545] The standard calibration curve is obtained by adding graded concentrations of KNO₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) into the calibration solution containing KI and H₂SO₄. The specificity of the Iso-NO electrode to NO is previously determined by measurement of NO from authentic NO gas (1050). The culture medium is removed and HUVECs are washed twice with Dulbecco's phosphate buffered saline. The cells are then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-well plates, and the cell plates are kept on a slide warmer (Lab Line Instruments Inc.) To maintain the temperature at 37° C. The NO sensor probe is inserted vertically into the wells, keeping the tip of the electrode 2 mm under the surface of the solution, before addition of the different conditions. S-nitroso acetyl penicillamin (SNAP) is used as a positive control. The amount of released NO is expressed as picomoles per 1×10⁶ endothelial cells. All values reported are means of four to six measurements in each group (number of cell culture wells). See, Leak et al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

[1546] The studies described in this example tested activity of polypeptides of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 41

[1547] Effect of Polypepides of the Invention on Cord Formation in Angiogenesis

[1548] Another step in angiogenesis is cord formation, marked by differentiation of endothelial cells. This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (hollow structures) when cultured in vitro.

[1549] CADMEC (microvascular endothelial cells) are purchased from Cell Applications, Inc. as proliferating (passage 2) cells and are cultured in Cell Applications' CADMEC Growth Medium and used at passage 5. For the in vitro angiogenesis assay, the wells of a 48-well cell culture plate are coated with Cell Applications' Attachment Factor Medium (200 ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wells at 7,500 cells/well and cultured overnight in Growth Medium. The Growth Medium is then replaced with 300 mg Cell Applications° Chord Formation Medium containing control buffer or a polypeptide of the invention (0.1 to 100 ng/ml) and the cells are cultured for an additional 48 hr. The numbers and lengths of the capillary-like chords are quantitated through use of the Boeckeler VIA-170 video image analyzer. All assays are done in triplicate.

[1550] Commercial (R&D) VEGF (50 ng/ml) is used as a positive control. b-esteradiol (1 ng/ml) is used as a negative control. The appropriate buffer (without protein) is also utilized as a control.

[1551] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 42

[1552] Angiogenic Effect on Chick Chorioallantoic Membrane

[1553] Chick chorioallantoic membrane (CAM) is a well-established system to examine angiogenesis. Blood vessel formation on CAM is easily visible and quantifiable. The ability of polypeptides of the invention to stimulate angiogenesis in CAM can be examined.

[1554] Fertilized eggs of the White Leghorn chick (Gallus gallus) and the Japanese qual (Coturnix coturnix) are incubated at 37.8° C. and 80% humidity. Differentiated CAM of 16-day-old chick and 13-day-old qual embryos is studied with the following methods.

[1555] On Day 4 of development, a window is made into the egg shell of chick eggs. The embryos are checked for normal development and the eggs sealed with cellotape. They are further incubated until Day 13. Thermanox coverslips (Nunc, Naperville, Ill.) are cut into disks of about 5 mm in diameter. Sterile and salt-free growth factors are dissolved in distilled water and about 3.3 mg/5 ml are pipetted on the disks. After air-drying, the inverted disks are applied on CAM. After 3 days, the specimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsed in 0.12 M sodium cacodylate buffer. They are photographed with a stereo microscope [Wild M8] and embedded for semi-and ultrathin sectioning as described above. Controls are performed with carrier disks alone.

[1556] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 43

[1557] Angiogenesis Assay Using a Matrigel Implant in Mouse

[1558] In vivo angiogenesis assay of a polypeptide of the invention measures the ability of an existing capillary network to form new vessels in an implanted capsule of murine extracellular matrix material (Matrigel). The protein is mixed with the liquid Matrigel at 4 degree C. and the mixture is then injected subcutaneously in mice where it solidifies. After 7 days, the solid “plug” of Matrigel is removed and examined for the presence of new blood vessels. Matrigel is purchased from Becton Dickinson Labware/Collaborative Biomedical Products.

[1559] When thawed at 4 degree C. the Matrigel material is a liquid. The Matrigel is mixed with a polypeptide of the invention at 150 ng/ml at 4 degrees C. and drawn into cold 3 ml syringes. Female C57B⅙ mice approximately 8 weeks old are injected with the mixture of Matrigel and experimental protein at 2 sites at the midventral aspect of the abdomen (0.5 ml/site). After 7 days, the mice are sacrificed by cervical dislocation, the Matrigel plugs are removed and cleaned (i.e., all clinging membranes and fibrous tissue is removed). Replicate whole plugs are fixed in neutral buffered 10% formaldehyde, embedded in paraffin and used to produce sections for histological examination after staining with Masson's Trichrome. Cross sections from 3 different regions of each plug are processed. Selected sections are stained for the presence of vWF. The positive control for this assay is bovine basic FGF (150 ng/ml). Matrigel alone is used to determine basal levels of angiogenesis.

[1560] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 44

[1561] Rescue of Ischemia in Rabbit Lower Limb Model

[1562] To study the in vivo effects of polynucleotides and polypeptides of the invention on ischemia, a rabbit hindlimb ischemia model is created by surgical removal of one femoral arteries as described previously (Takeshita et al., Am J. Pathol 147:1649-1660 (1995)). The excision of the femoral artery results in retrograde propagation of thrombus and occlusion of the external iliac artery. Consequently, blood flow to the ischemic limb is dependent upon collateral vessels originating from the internal iliac artery (Takeshitaet al. Am J. Pathol 147:1649-1660 (1995)). An interval of 10 days is allowed for post-operative recovery of rabbits and development of endogenous collateral vessels. At 10 day post-operatively (day 0), after performing a baseline angiogram, the internal iliac artery of the ischemic limb is transfected with 500 mg naked expression plasmid containing a polynucleotide of the invention by arterial gene transfer technology using a hydrogel-coated balloon catheter as described (Riessen et al. Hum Gene Ther. 4:749-758 (1993); Leclerc et al. J. Clin. Invest. 90: 936-944 (1992)). When a polypeptide of the invention is used in the treatment, a single bolus of 500 mg polypeptide of the invention or control is delivered into the internal iliac artery of the ischemic limb over a period of 1 min. through an infusion catheter. On day 30, various parameters are measured in these rabbits: (a) BP ratio—The blood pressure ratio of systolic pressure of the ischemic limb to that of normal limb; (b) Blood Flow and Flow Reserve—Resting FL: the blood flow during undilated condition and Max FL: the blood flow during fully dilated condition (also an indirect measure of the blood vessel amount) and Flow Reserve is reflected by the ratio of max FL: resting FL; (c) Angiographic Score—This is measured by the angiogram of collateral vessels. A score is determined by the percentage of circles in an overlaying grid that with crossing opacified arteries divided by the total number m the rabbit thigh; (d) Capillary density—The number of collateral capillaries determined in light microscopic sections taken from hindlimbs.

[1563] The studies described in this example tested activity of polynucleotides and polypeptides of the invention. However, one skilled in the art could easily modify the exemplified studies to test the agonists, and/or antagonists of the invention.

Example 45

[1564] Effect of Polypeptides of the Invention on Vasodilation

[1565] Since dilation of vascular endothelium is important in reducing blood pressure, the ability of polypeptides of the invention to affect the blood pressure in spontaneously hypertensive rats (SHR) is examined. Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of the polypeptides of the invention are administered to 13-14 week old spontaneously hypertensive rats (SHR). Data are expressed as the mean ±SEM. Statistical analysis are performed with a paired t-test and statistical significance is defined as p<0.05 vs. the response to buffer alone.

[1566] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 46

[1567] Rat Ischemic Skin Flap Model

[1568] The evaluation parameters include skin blood flow, skin temperature, and factor VIII immunohistochemistry or endothelial alkaline phosphatase reaction. Expression of polypeptides of the invention, during the skin ischemia, is studied using in situ hybridization.

[1569] The study in this model is divided into three parts as follows:

[1570] Ischemic skin

[1571] Ischemic skin wounds

[1572] Normal wounds

[1573] The experimental protocol includes:

[1574] Raising a 3×4 cm, single pedicle full-thickness random skin flap (myocutaneous flap over the lower back of the animal).

[1575] An excisional wounding (4-6 mm in diameter) in the ischemic skin (skin-flap).

[1576] Topical treatment with a polypeptide of the invention of the excisional wounds (day 0, 1, 2, 3, 4 post-wounding) at the following various dosage ranges: 1 mg to 100 mg.

[1577] Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21 post-wounding for histological, immunohistochemical, and in situ studies.

[1578] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 47

[1579] Peripheral Arterial Disease Model

[1580] Angiogenic therapy using a polypeptide of the invention is a novel therapeutic strategy to obtain restoration of blood flow around the ischemia in case of peripheral arterial diseases. The experimental protocol includes:

[1581] One side of the femoral artery is ligated to create ischemic muscle of the hindlimb, the other side of hindlimb serves as a control.

[1582] A polypeptide of the invention, in a dosage range of 20 mg-500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-3 weeks.

[1583] The ischemic muscle tissue is collected after ligation of the femoral artery at 1, 2, and 3 weeks for the analysis of expression of a polypeptide of the invention and histology. Biopsy is also performed on the other side of normal muscle of the contralateral hindlimb.

[1584] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 48

[1585] Ischemic Myocardial Disease Model

[1586] A polypeptide of the invention is evaluated as a potent mitogen capable of stimulating the development of collateral vessels, and restructuring new vessels after coronary artery occlusion. Alteration of expression of the polypeptide is investigated in situ. The experimental protocol includes:

[1587] The heart is exposed through a left-side thoracotomy in the rat. Immediately, the left coronary artery is occluded with a thin suture (6-0) and the thorax is closed.

[1588] A polypeptide of the invention, in a dosage range of 20 mg-500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-4 weeks.

[1589] Thirty days after the surgery, the heart is removed and cross-sectioned for morphometric and in situ analyzes.

[1590] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 49

[1591] Rat Corneal Wound Healing Model

[1592] This animal model shows the effect of a polypeptide of the invention on neovascularization. The experimental protocol includes:

[1593] Making a 1-1.5 mm long incision from the center of cornea into the stromal layer. Inserting a spatula below the lip of the incision facing the outer corner of the eye. Making a pocket (its base is 1-1.5 mm form the edge of the eye). Positioning a pellet, containing 50 ng-5 ug of a polypeptide of the invention, within the pocket.

[1594] Treatment with a polypeptide of the invention can also be applied topically to the corneal wounds in a dosage range of 20 mg-500 mg (daily treatment for five days).

[1595] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 50

[1596] Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models

[1597] Diabetic db+/db+ Mouse Model.

[1598] To demonstrate that a polypeptide of the invention accelerates the healing process, the genetically diabetic mouse model of wound healing is used. The full thickness wound healing model in the db+/db+ mouse is a well characterized, clinically relevant and reproducible model of impaired wound healing. Healing of the diabetic wound is dependent on formation of granulation tissue and re-epithelialization rather than contraction (Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).

[1599] The diabetic animals have many of the characteristic features observed in Type II diabetes mellitus. Homozygous (db+/db+) mice are obese in comparison to their normal heterozygous (db+/+m) littermates. Mutant diabetic (db+/db+) mice have a single autosomal recessive mutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Mandel et al., J. Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol. 51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular lesions, basement membrane thickening and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes 29(1):60-67 (1980); Giacomelli et Lab Invest. 40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl): 1-6 (1982)). These homozygous diabetic mice develop hyperglycemia that is resistant to insulin analogous to human type II diabetes (Mandel et al., J. Immunol. 120:1375-1377 (1978)).

[1600] The characteristics observed in these animals suggests that healing in this model may be similar to the healing observed in human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

[1601] Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic (db+/+m) heterozygous littermates are used in this study (Jackson Laboratories). The animals are purchased at 6 weeks of age and are 8 weeks old at the beginning of the study. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. The experiments are conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.

[1602] Wounding protocol is performed according to previously reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)). Briefly, on the day of wounding, animals are anesthetized with an intraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal region of the animal is shaved and the skin washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is then created using a Keyes tissue punch. Immediately following wounding, the surrounding skin is gently stretched to eliminate wound expansion. The wounds are left open for the duration of the experiment. Application of the treatment is given topically for 5 consecutive days commencing on the day of wounding. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.

[1603] Wounds are visually examined and photographed at a fixed distance at the day of surgery and at two day intervals thereafter. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

[1604] A polypeptide of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.

[1605] Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

[1606] Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3) treated group.

[1607] Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total square area of the wound. Contraction is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm², the corresponding size of the dermal punch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

[1608] Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds are used to assess whether the healing process and the morphologic appearance of the repaired skin is altered by treatment with a polypeptide of the invention. This assessment included verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometer is used by a blinded observer.

[1609] Tissue sections are also stained immunohistochemically with a polyclonal rabbit anti-human keratin antibody using ABC Elite detection system. Human skin is used as a positive tissue control while non-immune IgG is used as a negative control. Keratinocyte growth is determined by evaluating the extent of reepithelialization of the wound using a calibrated lens micrometer.

[1610] Proliferating cell nuclear antigen/cyclin (PCNA), in skin specimens is demonstrated by using anti-PCNA antibody (1:50) with an ABC Elite detection system. Human colon cancer can serve as a positive tissue control and human brain tissue can be used as a negative tissue control. Each specimen includes a section with omission of the primary antibody and substitution with non-immune mouse IgG. Ranking of these sections is based on the extent of proliferation on a scale of 0-8, the lower side of the scale reflecting slight proliferation to the higher side reflecting intense proliferation.

[1611] Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.

[1612] Steroid Impaired Rat Model

[1613] The inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989); Wahlet al., J. Immunol. 115: 476-481 (1975); Werb et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retard wound healing by inhibiting angiogenesis, decreasing vascular permeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)), fibroblast proliferation, and collagen synthesis (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978)) and producing a transient reduction of circulating monocytes (Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989)). The systemic administration of steroids to impaired wound healing is a well establish phenomenon in rats (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989)).

[1614] To demonstrate that a polypeptide of the invention can accelerate the healing process, the effects of multiple topical applications of the polypeptide on full thickness excisional skin wounds in rats in which healing has been impaired by the systemic administration of methylprednisolone is assessed.

[1615] Young adult male Sprague Dawley rats weighing 250-300 g (Charles River Laboratories) are used in this example. The animals are purchased at 8 weeks of age and are 9 weeks old at the beginning of the study. The healing response of rats is impaired by the systemic administration of methylprednisolone (17 mg/kg/rat intramuscularly) at the time of wounding. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. This study is conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.

[1616] The wounding protocol is followed according to section A, above. On the day of wounding, animals are anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved and the skin washed with 70% ethanol and iodine solutions. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is created using a Keyes tissue punch. The wounds are left open for the duration of the experiment. Applications of the testing materials are given topically once a day for 7 consecutive days commencing on the day of wounding and subsequent to methylprednisolone administration. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.

[1617] Wounds are visually examined and photographed at a fixed distance at the day of wounding and at the end of treatment. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

[1618] The polypeptide of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.

[1619] Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

[1620] Four groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebo control 3) treated groups.

[1621] Wound closure is analyzed by measuring the area in-the vertical and horizontal axis and obtaining the total area of the wound. Closure is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm², the corresponding size of the dermal punch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

[1622] Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds allows assessment of whether the healing process and the morphologic appearance of the repaired skin is improved by treatment with a polypeptide of the invention. A calibrated lens micrometer is used by a blinded observer to determine the distance of the wound gap.

[1623] Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.

[1624] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 51

[1625] Lymphadema Animal Model

[1626] The purpose of this experimental approach is to create an appropriate and consistent lymphedema model for testing the therapeutic effects of a polypeptide of the invention in lymphangiogenesis and re-establishment of the lymphatic circulatory system in the rat hind limb. Effectiveness is measured by swelling volume of the affected limb, quantification of the amount of lymphatic vasculature, total blood plasma protein, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progress of the edema is followed for up to 3-4 weeks.

[1627] Prior to beginning surgery, blood sample is drawn for protein concentration analysis. Male rats weighing approximately ˜350 g are dosed with Pentobarbital. Subsequently, the right legs are shaved from knee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. Circumference and volumetric measurements are made prior to injecting dye into paws after marking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of both right and left paws are injected with 0.05 ml of 1% Evan's Blue. Circumference and volumetric measurements are then made following injection of dye into paws.

[1628] Using the knee joint as a landmark, a mid-leg inguinal incision is made circumferentially allowing the femoral vessels to be located. Forceps and hemostats are used to dissect and separate the skin flaps. After locating the femoral vessels, the lymphatic vessel that runs along side and underneath the vessel(s) is located. The main lymphatic vessels in this area are then electrically coagulated suture ligated.

[1629] Using a microscope, muscles in back of the leg (near the semitendinosis and adductors) are bluntly dissected. The popliteal lymph node is then located. The 2 proximal and 2 distal lymphatic vessels and distal blood supply of the popliteal node are then and ligated by suturing. The popliteal lymph node, and any accompanying adipose tissue, is then removed by cutting connective tissues.

[1630] Care is taken to control any mild bleeding resulting from this procedure. After lymphatics are occluded, the skin flaps are sealed by using liquid skin (Vetbond) (A J Buck). The separated skin edges are sealed to the underlying muscle tissue while leaving a gap of ˜0.5 cm around the leg. Skin also may be anchored by suturing to underlying muscle when necessary.

[1631] To avoid infection, animals are housed individually with mesh (no bedding). Recovering animals are checked daily through the optimal edematous peak, which typically occurred by day 5-7. The plateau edematous peak are then observed. To evaluate the intensity of the lymphedema, the circumference and volumes of 2 designated places on each paw before operation and daily for 7 days are measured. The effect plasma proteins on lymphedema is determined and whether protein analysis is a useful testing perimeter is also investigated. The weights of both control and edematous limbs are evaluated at 2 places. Analysis is performed in a blind manner.

[1632] Circumference Measurements: Under brief gas anesthetic to prevent limb movement, a cloth tape is used to measure limb circumference. Measurements are done at the ankle bone and dorsal paw by 2 different people then those 2 readings are averaged. Readings are taken from both control and edematous limbs.

[1633] Volumetric Measurements: On the day of surgery, animals are anesthetized with Pentobarbital and are tested prior to surgery. For daily volumetrics animals are under brief halothane anesthetic (rapid immobilization and quick recovery), both legs are shaved and equally marked using waterproof marker on legs. Legs are first dipped in water, then dipped into instrument to each marked level then measured by Buxco edema software(Chen/Victor). Data is recorded by one person, while the other is dipping the limb to marked area.

[1634] Blood-plasma protein measurements: Blood is drawn, spun, and serum separated prior to surgery and then at conclusion for total protein and Ca2+ comparison.

[1635] Limb Weight Comparison: After drawing blood, the animal is prepared for tissue collection. The limbs are amputated using a quillitine, then both experimental and control legs are cut at the ligature and weighed. A second weighing is done as the tibio-cacaneal joint is disarticulated and the foot is weighed.

[1636] Histological Preparations: The transverse muscle located behind the knee (popliteal) area is dissected and arranged in a metal mold, filled with freezeGel, dipped into cold methylbutane, placed into labeled sample bags at −80 EC until sectioning. Upon sectioning, the muscle is observed under fluorescent microscopy for lymphatics..

[1637] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 52

[1638] Suppression of TNF Alpha-Induced Adhesion Molecule Expression by a Polypeptide of the Invention

[1639] The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.

[1640] Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine, is a stimulator of all three CAMs on endothelial cells and may be involved in a wide variety of inflammatory responses, often resulting in a pathological outcome.

[1641] The potential of a polypeptide of the invention to mediate a suppression of TNF-a induced CAM expression can be examined. A modified ELISA assay which uses ECs as a solid phase absorbent is employed to measure the amount of CAM expression on TNF-a treated ECs when co-stimulated with a member of the FGF family of proteins.

[1642] To perform the experiment, human umbilical vein endothelial cell (HUVEC) cultures are obtained from pooled cord harvests and maintained in growth medium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCS and 1% penicillin/streptomycin in a 37 degree C. humidified incubator containing 5% CO₂. HUVECs are seeded in 96-well plates at concentrations of 1×10⁴ cells/well in EGM medium at 37 degree C. for 18-24 hrs or until confluent. The monolayers are subsequently washed 3 times with a serum-free solution of RPMI-1640 supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin, and treated with a given cytokine and/or growth factor(s) for 24 h at 37 degree C. Following incubation, the cells are then evaluated for CAM expression.

[1643] Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard 96 well plate to confluence. Growth medium is removed from the cells and replaced with 90 ul of 199 Medium (10% FBS). Samples for testing and positive or negative controls are added to the plate in triplicate (in 10 ul volumes). Plates are incubated at 37 degree C. for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS(with Ca++ and Mg++) is added to each well. Plates are held at 4° C. for 30 min.

[1644] Fixative is then removed from the wells and wells are washed 1× with PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10 μl of diluted primary antibody to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37° C. for 30 min. in a humidified environment. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA.

[1645] Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphotase (1:5,000 dilution) to each well and incubated at 37° C. for 30 min. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol Phosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000 (10°)>10⁻⁰ ⁵>10⁻¹>10^(−1.5)0.5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added to each of the standard wells. The plate must be incubated at 37° C. for 4 h. A volume of 50 μl of 3M NaOH is added to all wells. The results are quantified on a plate reader at 405 nm. The background subtraction option is used on blank wells filled with glycine buffer only. The template is set up to indicate the concentration of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.

[1646] The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 53

[1647] Assay for the Stimulation of Bone Marrow CD34+ Cell Proliferation

[1648] This assay is based on the ability of human CD34+ to proliferate in the presence of hematopoietic growth factors and evaluates the ability of isolated polypeptides expressed in mammalian cells to stimulate proliferation of CD34+ cells.

[1649] It has been previously shown that most mature precursors will respond to only a single signal. More immature precursors require at least two signals to respond. Therefore, to test the effect of polypeptides on hematopoietic activity of a wide range of progenitor cells, the assay contains a given polypeptide in the presence or absence of other hematopoietic growth factors. Isolated cells are cultured for 5 days in the presence of Stem Cell Factor (SCF) in combination with tested sample. SCF alone has a very limited effect on the proliferation of bone marrow (BM) cells, acting in such conditions only as a “survival” factor. However, combined with any factor exhibiting stimulatory effect on these cells (e.g., IL-3), SCF will cause a synergistic effect. Therefore, if the tested polypeptide has a stimulatory effect on a hematopoietic progenitors, such activity can be easily detected. Since normal BM cells have a low level of cycling cells, it is likely that any inhibitory effect of a given polypeptide, or agonists or antagonists thereof, might not be detected. Accordingly, assays for an inhibitory effect on progenitors is preferably tested in cells that are first subjected to in vitro stimulation with SCF+IL+3, and then contacted with the compound that is being evaluated for inhibition of such induced proliferation.

[1650] Briefly, CD34+ cells are isolated using methods known in the art. The cells are thawed and resuspended in medium (QBSF 60 serum-free medium with 1% L-glutamine (500 ml) Quality Biological, Inc., Gaithersburg, Md. Cat#160-204-101). After several gentle centrifugation steps at 200×g, cells are allowed to rest for one hour. The cell count is adjusted to 2.5×10⁵ cells/ml. During this time, 100 μl of sterile water is added to the peripheral wells of a 96-well plate. The cytokines that can be tested with a given polypeptide in this assay is rhSCF (R&D Systems, Minneapolis, Minn., Cat#255-SC) at 50 ng/ml alone and in combination with rhSCF and rhIL-3 (R&D Systems, Minneapolis, Minn., Cat#203-ML) at 30 ng/ml. After one hour, 10 μl of prepared cytokines, 50 μl SID (supernatants at 1:2 dilution=50 μl) and 20 μl of diluted cells are added to the media which is already present in the wells to allow for a final total volume of 100 μl. The plates are then placed in a 37° C./15% CO₂ incubator for five days.

[1651] Eighteen hours before the assay is harvested, 0.5 μCi/well of [3H] Thymidine is added in a 10 μl volume to each well to determine the proliferation rate. The experiment is terminated by harvesting the cells from each 96-well plate to a filtermat using the Tomtec Harvester 96. After harvesting, the filtermats are dried, trimmed and placed into OmniFilter assemblies consisting of one OmniFilter plate and one OmniFilter Tray. 60 μl Microscint is added to each well and the plate sealed with TopSeal-A press-on sealing film A bar code 15 sticker is affixed to the first plate for counting. The sealed plates is then loaded and the level of radioactivity determined via the Packard Top Count and the printed data collected for analysis. The level of radioactivity reflects the amount of cell proliferation.

[1652] The studies described in this example test the activity of a given polypeptide to stimulate bone marrow CD34+ cell proliferation. One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof. As a nonlimiting example, potential antagonists tested in this assay would be expected to inhibit cell proliferation in the presence of cytokines and/or to increase the inhibition of cell proliferation in the presence of cytokines and a given polypeptide. In contrast, potential agonists tested in this assay would be expected to enhance cell proliferation and/or to decrease the inhibition of cell proliferation in the presence of cytokines and a given polypeptide.

[1653] The ability of a gene to stimulate the proliferation of bone marrow CD34+ cells indicates that polynucleotides and polypeptides corresponding to the gene are useful for the diagnosis and treatment of disorders affecting the immune system and hematopoiesis. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections above, and elsewhere herein.

Example 54

[1654] Assay for Extracellular Matrix Enhanced Cell Response (EMECR)

[1655] The objective of the Extracellular Matrix Enhanced Cell Response (EMECR) assay is to identify gene products (e.g., isolated polypeptides) that act on the hematopoietic stem cells in the context of the extracellular matrix (ECM) induced signal.

[1656] Cells respond to the regulatory factors in the context of signal(s) received from the surrounding microenvironment. For example, fibroblasts, and endothelial and epithelial stem cells fail to replicate in the absence of signals from the ECM. Hematopoietic stem cells can undergo self-renewal in the bone marrow, but not in in vitro suspension culture. The ability of stem cells to undergo self-renewal in vitro is dependent upon their interaction with the stromal cells and the ECM protein fibronectin (fn). Adhesion of cells to fn is mediated by the α₅.β₁ and α₄.β₁ integrin receptors, which are expressed by human and mouse hematopoietic stem cells. The factor(s) which integrate with the ECM environment and responsible for stimulating stem cell self-renewal has not yet been identified. Discovery of such factors should be of great interest in gene therapy and bone marrow transplant applications

[1657] Briefly, polystyrene, non tissue culture treated, 96-well plates are coated with fn fragment at a coating concentration of 0.2 μg/cm². Mouse bone marrow cells are plated (1,000 cells/well ) in 0.2 ml of serum-free medium. Cells cultured in the presence of IL-3 (5 ng/ml )+SCF (50 ng/ml) would serve as the positive control, conditions under which little self-renewal but pronounced differentiation of the stem cells is to be expected. Gene products are tested with appropriate negative controls in the presence and absence of SCF(5.0 ng/ml), where test factor supernates represent 10% of the total assay volume. The plated cells are then allowed to grow by incubating in a low oxygen environment (5% CO₂, 7% O₂, and 88% N₂ ) tissue culture incubator for 7 days. The number of proliferating cells within the wells is then quantitated by measuring thymidine incorporation into cellular DNA. Verification of the positive hits in the assay will require phenotypic characterization of the cells, which can be accomplished by scaling up of the culture system and using appropriate antibody reagents against cell surface antigens and FACScan.

[1658] One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.

[1659] If a particular gene product is found to be a stimulator of hematopoietic progenitors, polynucleotides and polypeptides corresponding to the gene may be useful for the diagnosis and treatment of disorders affecting the immune system and hematopoiesis. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections above, and elsewhere herein. The gene product may also be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.

[1660] Additionally, the polynucleotides and/or polypeptides of the gene of interest and/or agonists and/or antagonists thereof, may also be employed to inhibit the proliferation and differentiation of hematopoietic cells and therefore may be employed to protect bone marrow stem cells from chemotherapeutic agents during chemotherapy. This antiproliferative effect may allow administration of higher doses of chemotherapeutic agents and, therefore, more effective chemotherapeutic treatment.

[1661] Moreover, polynucleotides and polypeptides corresponding to the gene of interest may also be useful for the treatment and diagnosis of hematopoietic related disorders such as, for example, anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.

Example 55

[1662] Human Dermal Fibroblast and Aortic Smooth Muscle Cell Proliferation

[1663] The polypeptide of interest is added to cultures of normal human dermal fibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC) and two co-assays are performed with each sample. The first assay examines the effect of the polypeptide of interest on the proliferation of normal human dermal fibroblasts (NHDF) or aortic smooth muscle cells (AoSMC). Aberrant growth of fibroblasts or smooth muscle cells is a part of several pathological processes, including fibrosis, and restenosis. The second assay examines IL6 production by both NHDF and SMC. IL6 production is an indication of functional activation. Activated cells will have increased production of a number of cytokines and other factors, which can result in a proinflammatory or immunomodulatory outcome. Assays are run with and without co-TNFa stimulation, in order to check for costimulatory or inhibitory activity.

[1664] Briefly, on day 1, 96-well black plates are set up with 1000 cells/well (NHDF) or 2000 cells/well (AoSMC) in 100 μl culture media. NHDF culture media contains: Clonetics FB basal media, 1 mg/ml hFGF, 5 mg/ml insulin, 50 mg/ml gentamycin, 2% FBS, while AoSMC culture media contains Clonetics SM basal media, 0.5 μg/ml hEGF, 5 mg/ml insulin, 1 μg/ml hFGF, 50 mg/ml gentamycin, 50 μg/ml Amphotericin B, 5% FBS. After incubation @ 37° C. for at least 4-5 hours culture media is aspirated and replaced with growth arrest media. Growth arrest media for NHDF contains fibroblast basal media, 50 mg/ml gentamycin, 2% FBS, while growth arrest media for AoSMC contains SM basal media, 50 mg/ml gentamycin, 50 μg/ml Amphotericin B, 0.4% FBS. Incubate at 37 C. until day 2.

[1665] On day 2, serial dilutions and templates of the polypeptide of interest are designed which should always include media controls and known-protein controls. For both stimulation and inhibition experiments, proteins are diluted in growth arrest media. For inhibition experiments, TNFa is added to a final concentration of 2 ng/ml (NHDF) or 5 ng/ml (AoSMC). Then add ⅓ vol media containing controls or supernatants and incubate at 37 C./5% CO₂ until day 5.

[1666] Transfer 60 μl from each well to another labeled 96-well plate, cover with a plate-sealer, and store at 4 C. until Day 6 (for IL6 ELISA). To the remaining 100 μl in the cell culture plate, aseptically add Alamar Blue in an amount equal to 10% of the culture volume (10 μl). Return plates to incubator for 3 to 4 hours. Then measure fluorescence with excitation at 530 nm and emission at 590 nm using the CytoFluor. This yields the growth stimulation/inhibition data.

[1667] On day 5, the IL6 ELISA is performed by coating a 96 well plate with 50-100 ul/well of Anti-Human IL6 Monoclonal antibody diluted in PBS, pH 7.4, incubate ON at room temperature.

[1668] On day 6, empty the plates into the sink and blot on paper towels. Prepare Assay Buffer containing PBS with 4% BSA. Block the plates with 200 μl/well of Pierce Super Block blocking buffer in PBS for 1-2 hr and then wash plates with wash buffer (PBS, 0.05% Tween-20). Blot plates on paper towels. Then add 50 μl/well of diluted Anti-Human IL-6 Monoclonal, Biotin-labeled antibody at 0.50 mg/ml. Make dilutions of IL-6 stock in media (30, 10, 3, 1, 0.3, 0 ng/ml). Add duplicate samples to top row of plate. Cover the plates and incubate for 2 hours at RT on shaker.

[1669] Wash plates with wash buffer and blot on paper towels. Dilute EU-labeled Streptavidin 1:1000 in Assay buffer, and add 100 μl/well. Cover the plate and incubate 1 h at RT. Wash plates with wash buffer. Blot on paper towels.

[1670] Add 100 μl/well of Enhancement Solution. Shake for 5 minutes. Read the plate on the Wallac DELFIA Fluorometer. Readings from triplicate samples in each assay were tabulated and averaged.

[1671] A positive result in this assay suggests AoSMC cell proliferation and that the gene product of interest may be involved in dermal fibroblast proliferation and/or smooth muscle cell proliferation. A positive result also suggests many potential uses of polypeptides, polynucleotides, agonists and/or antagonists of the gene/gene product of interest. For example, inflammation and immune responses, wound healing, and angiogenesis, as detailed throughout this specification. Particularly, polypeptides of the gene product and polynucleotides of the gene may be used in wound healing and dermal regeneration, as well as the promotion of vasculargenesis, both of the blood vessels and lymphatics. The growth of vessels can be used in the treatment of, for example, cardiovascular diseases. Additionally, antagonists of polypeptides of the gene product and polynucleotides of the gene may be useful in treating diseases, disorders, and/or conditions which involve angiogenesis by acting as an anti-vascular (e.g., anti-angiogenesis). These diseases, disorders, and/or conditions are known in the art and/or are described herein, such as, for example, malignancies, solid tumors, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis. Moreover, antagonists of polypeptides of the gene product and polynucleotides of the gene may be useful in treating anti-hyperproliferative diseases and/or anti-inflammatory known in the art and/or described herein.

[1672] One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.

Example 56

[1673] Cellular Adhesion Molecule (CAM) Expression on Endothelial Cells

[1674] The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.

[1675] Briefly, endothelial cells (e.g., Human Umbilical Vein Endothelial cells (HUVECs)) are grown in a standard 96 well plate to confluence, growth medium is removed from the cells and replaced with 100 μl of 199 Medium (10% fetal bovine serum (FBS)). Samples for testing and positive or negative controls are added to the plate in triplicate (in 10 μl volumes). Plates are then incubated at 37° C. for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 μl of 0.1% parafo r maldehyde-PBS(with Ca++ and Mg++) is added to each well. Plates are held at 4° C. for 30 min. Fixative is removed from the wells and wells are washed 1× with PBS(+Ca,Mg)+0.5% BSA and drained. 10 μl of diluted primary antibody is added to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37° C. for 30 min. in a humidified environment. Wells are washed three times with PBS(+Ca,Mg)+0.5% BSA. 20 μl of diluted ExtrAvidin-Alkaline Phosphotase (1:5,000 dilution, refered to herein as the working dilution) are added to each well and incubated at 37° C. for 30 min. Wells are washed three times with PBS(+Ca,Mg)+0.5% BSA. Dissolve 1 tablet of p-Nitrophenol Phosphate pNPP per 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000 (10°)>10^(−0.5)>10⁻¹>10^(−1.5) 0.5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent is then added to each of the standard wells. The plate is incubated at 37° C. for 4h. A volume of 50 μl of 3M NaOH is added to all wells. The plate is read on a plate reader at 405 nm using the background subtraction option on blank wells filled with glycine buffer only. Additionally, the template is set up to indicate the concentration of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.

Example 57

[1676] Alamar Blue Endothelial Cells Proliferation Assay

[1677] This assay may be used to quantitatively determine protein mediated inhibition of bFGF-induced proliferation of Bovine Lymphatic Endothelial Cells (LECs), Bovine Aortic Endothelial Cells (BAECs) or Human Microvascular Uterine Myometrial Cells (UTMECs). This assay incorporates a fluorometric growth indicator based on detection of metabolic activity. A standard Alamar Blue Proliferation Assay is prepared in EGM-2MV with 10 ng/ml of bFGF added as a source of endothelial cell stimulation. This assay may be used with a variety of endothelial cells with slight changes in growth medium and cell concentration. Dilutions of the protein batches to be tested are diluted as appropriate. Serum-free medium (GIBCO SFM) without bFGF is used as a non-stimulated control and Angiostatin or TSP-1 are included as a known inhibitory controls.

[1678] Briefly, LEC, BAECs or UTMECs are seeded in growth media at a density of 5000 to 2000 cells/well in a 96 well plate and placed at 37-C. overnight. After the overnight incubation of the cells, the growth media is removed and replaced with GIBCO EC-SFM. The cells are treated with the appropriate dilutions of the protein of interest or control protein sample(s) (prepared in SFM) in triplicate wells with additional bFGF to a concentration of 10 ng/ml. Once the cells have been treated with the samples, the plate(s) is/are placed back in the 37° C. incubator for three days. After three days 10 ml of stock alamar blue (Biosource Cat#DAL1100) is added to each well and the plate(s) is/are placed back in the 37° C. incubator for four hours. The plate(s) are then read at 530 nm excitation and 590 nm emission using the CytoFluor fluorescence reader. Direct output is recorded in relative fluorescence units.

[1679] Alamar blue is an oxidation-reduction indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth. As cells grow in culture, innate metabolic activity results in a chemical reduction of the immediate surrounding environment. Reduction related to growth causes the indicator to change from oxidized (non-fluorescent blue) form to reduced (fluorescent red) form. i.e. stimulated proliferation will produce a stronger signal and inhibited proliferation will produce a weaker signal and the total signal is proportional to the total number of cells as well as their metabolic activity. The background level of activity is observed with the starvation medium alone. This is compared to the output observed from the positive control samples (bFGF in growth medium) and protein dilutions.

Example 58

[1680] Detection of Inhibition of a Mixed Lymphocyte Reaction

[1681] This assay can be used to detect and evaluate inhibition of a Mixed Lymphocyte Reaction (MLR) by gene products (e.g., isolated polypeptides). Inhibition of a MLR may be due to a direct effect on cell proliferation and viability, modulation of costimulatory molecules on interacting cells, modulation of adhesiveness between lymphocytes and accessory cells, or modulation of cytokine production by accessory cells. Multiple cells may be targeted by these polypeptides since the peripheral blood mononuclear fraction used in this assay includes T, B and natural killer lymphocytes, as well as monocytes and dendritic cells.

[1682] Polypeptides of interest found to inhibit the MLR may find application in diseases associated with lymphocyte and monocyte activation or proliferation. These include, but are not limited to, diseases such as asthma, arthritis, diabetes, inflammatory skin conditions, psoriasis, eczema, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, inflammatory bowel disease, crohn's disease, ulcerative colitis, arteriosclerosis, cirrhosis, graft vs. host disease, host vs. graft disease, hepatitis, leukemia and lymphoma.

[1683] Briefly, PBMCs from human donors are purified by density gradient centrifugation using Lymphocyte Separation Medium (LSM®, density 1.0770 g/ml, Organon Teknika Corporation, West Chester, Pa.). PBMCs from two donors are adjusted to 2×10⁶ cells/ml in RPMI-1640 (Life Technologies, Grand Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine. PBMCs from a third donor is adjusted to 2×10⁵ cells/ml. Fifty microliters of PBMCs from each donor is added to wells of a 96-well round bottom microtiter plate. Dilutions of test materials (50 μl) is added in triplicate to microtiter wells. Test samples (of the protein of interest) are added for final dilution of 1:4; rhuIL-2 (R&D Systems, Minneapolis, Minn., catalog number 202-IL) is added to a final concentration of 1 μg/ml; anti-CD4 mAb (R&D Systems, clone 34930.11, catalog number MAB379) is added to a final concentration of 10 μg/ml. Cells are cultured for 7-8 days at 37° C. in 5% CO₂, and 1 μC of [³H] thymidine is added to wells for the last 16 hrs of culture. Cells are harvested and thymidine incorporation determined using a Packard TopCount. Data is expressed as the mean and standard deviation of triplicate determinations.

[1684] Samples of the protein of interest are screened in separate experiments and compared to the negative control treatment, anti-CD4 mAb, which inhibits proliferation of lymphocytes and the positive control treatment, IL-2 (either as recombinant material or supernatant), which enhances proliferation of lymphocytes.

[1685] One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.

[1686] It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims. The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is hereby incorporated herein by reference. Further, the hard copy of the sequence listing submitted herewith and the corresponding computer readable form are both incorporated herein by reference in their entireties. Additionally, the contents of International Application No. PCT/US98/21142 and of Provisional Applications Serial Nos. 60/061,463, 60/061,529, 60/071,498, 60/061,527, 60/061,536 and 60/061,532 are all hereby incorporated by reference in their entirety. TABLE 6 Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 A . . B . . . −0.93 0.37 * * . −0.30 0.53 Lys 2 A . . B . . . −0.54 0.63 * * −0.60 0.34 Phe 3 A . . B . . . −1.04 0.20 * * . −0.30 0.45 Leu 4 A . . B . . . −1.47 0.46 * * . −0.60 0.32 Leu 5 A . . B . . . −1.89 0.53 * * . −0.60 0.13 Asp 6 A . . B . . . −2.10 1.21 * * . −0.60 0.12 Ile 7 A . . B . . . −2.96 1.11 * * . −0.60 0.12 Leu 8 A . . B . . . −2.47 1.11 . * . −0.60 0.12 Leu 9 A . . B . . . −2.47 0.86 . . . −0.60 0.11 Leu 10 A . . B . . . −2.47 1.54 . * . −0.60 0.14 Leu 11 A . . B . . . −3.36 1.54 . . . −0.60 0.14 Pro 12 A . . B . . . −3.32 1.54 . . . −0.60 0.11 Leu 13 A . . B . . . −3.18 1.50 . . . −0.60 0.10 Leu 14 . . B B . . . −2.67 1.39 . . . −0.60 0.07 Ile 15 . . B B . . . −2.67 1.09 . . . −0.60 0.06 Val 16 . . B B . . . −1.86 1.34 . . . −0.60 0.06 Cys 17 . . B B . . . −1.94 0.66 . . . −0.60 0.12 Ser 18 A . . B . . . −1.83 0.36 * . . −0.30 0.23 Leu 19 A A . . . . . −1.88 0.46 * * . −0.60 0.27 Glu 20 A A . B . . . −0.94 0.46 * * . −0.60 0.38 Ser 21 A A . B . . . −0.90 −0.11 * . . 0.30 0.56 Phe 22 A A . B . . . −0.93 0.19 * * . −0.30 0.56 Val 23 A A . B . . . −1.52 0.29 * * . −0.30 0.28 Lys 24 A A . B . . . −0.92 0.97 * * . −0.60 0.15 Leu 25 A A . B . . . −0.88 1.01 . * . −0.26 0.26 Phe 26 A A . B . . . −0.47 0.23 * . . 0.38 0.71 Ile 27 A A . B . . . 0.34 −0.41 * . . 1.32 0.70 Pro 28 A . . . . T . 1.24 −0.41 . * F 2.36 1.65 Lys 29 . . . . T T . 0.90 −1.10 . . F 3.40 3.81 Arg 30 . . . . T T . 0.86 −1.50 . . F 3.06 7.29 Arg 31 . . . . T T . 1.24 −1.54 . . F 2.81 3.50 Lys 32 . . . B T . . 1.79 −1.49 . . F 2.16 2.52 Ser 33 . . . B . . C 2.00 −1.06 . * F 1.71 1.28 Val 34 . . . B . . C 1.07 −1.06 . . F 1.46 1.13 Thr 35 . . B B . . . 0.10 −0.37 . * F 0.90 0.40 Gly 36 . . B B . . . −0.82 0.27 . * F 0.21 0.22 Glu 37 . . B B . . . −1.76 0.57 * . F −0.18 0.24 Ile 38 . . B B . . . −1.77 0.61 . * . −0.42 0.12 Val 39 . . B B . . . −1.26 0.61 . . . −0.51 0.17 Leu 40 . . B B . . . −1.53 0.61 . . . −0.60 0.10 Ile 41 . . B B . . −1.53 1.11 . . . −0.60 0.14 Thr 42 . . B B . . . −1.57 0.86 . . . −0.60 0.19 Gly 43 . . B . . T . −1.02 0.71 * . . −0.20 0.31 Ala 44 . . . . . T C −1.06 0.46 . . . 0.00 0.44 Gly 45 . . . . . T C −0.59 0.46 * * . 0.00 0.21 His 46 . . . . . T C 0.41 0.40 * * . 0.00 0.21 Gly 47 . . . . . . C −0.09 −0.03 * * . 0.70 0.42 Ile 48 . . B . . . . −0.06 0.16 * * . −0.10 0.35 Gly 49 . . B . . . . −0.06 0.21 * * . −0.10 0.37 Arg 50 . A B . . . . 0.04 0.21 * * . −0.30 0.37 Leu 51 A B . . . . 0.08 0.54 * * . −0.60 0.84 Thr 52 . A B . . . −0.28 −0.14 * . . 0.45 1.46 Ala 53 A A . . . . . 0.02 0.21 * * . −0.30 0.65 Tyr 54 A A . . . . . 0.41 0.71 * * . −0.60 0.79 Glu 55 A A . . . . . −0.51 0.03 * * . −0.15 1.10 Phe 56 A A . . . . . 0.34 0.23 . * . −0.30 0.90 Ala 57 A A . . . . . 0.36 −0.27 * * . 0.45 1.15 Lys 58 A A . . . . . 0.99 −0.64 * * F 0.75 0.89 Leu 59 A A . . . . . 0.42 −0.64 * * F 0.90 2.05 Lys 60 A A . . . . . −0.43 −0.74 * * F 0.90 1.67 Ser 61 A A . . . . . −0.54 −0.60 * * F 0.75 0.62 Lys 62 A A . . . . . −0.24 0.09 . * F −0.15 0.62 Leu 63 A A . . . . . −0.29 0.31 . * F −0.15 0.33 Val 64 A A . . . . . −0.37 0.31 . * . −0.30 0.41 Leu 65 A A . . . . . −0.41 0.61 * . . −0.60 0.14 Trp 66 A A . . . . . −0.07 1.01 * . . −0.60 0.28 Asp 67 A A . . . . . −0.14 0.33 * . . −0.30 0.75 Ile 68 A A . . . . . 0.32 0.19 * . . 0.12 1.23 Asn 69 A . . . . T . 0.37 −0.07 * . . 1.39 1.16 Lys 70 . . . . . T C 1.18 −0.30 * . F 1.86 0.57 His 71 . . . . . T C 1.47 −0.30 * . . 2.13 1.42 Gly 72 . . . . . T C 1.16 −0.99 * . . 2.70 1.53 Leu 73 A A . . . . . 1.46 −0.90 . . F 1.98 1.10 Glu 74 A A . . . . . 0.87 −0.40 . . F 1.26 0.82 Glu 75 A A . . . . . 0.87 −0.40 . . F 0.99 0.84 Thr 76 A A . . . . . 0.23 −0.83 * * F 1.17 2.03 Ala 77 A A . . . . . 0.62 −0.94 * F 0.75 0.63 Ala 78 A A . . . . . 1.09 −0.94 . . . 0.60 0.72 Lys 79 A A . . . . . 0.28 −0.51 . * F 0.75 0.50 Cys 80 A . . . . T . −0.07 −0.31 . * F 0.85 0.41 Lys 81 A . . . . T . −0.34 −0.39 . . F 0.85 0.40 Gly 82 A . . . . T . 0.29 −0.39 . * F 0.85 0.20 Leu 83 A . . . . T . 0.02 −0.39 * F 0.85 0.75 Gly 84 A . . . . . . −0.06 −0.31 . * . 0.50 0.28 Ala 85 A . . . . . . 0.30 0.19 * . . −0.10 0.38 Lys 86 A . . B . . . −0.44 0.24 * * . −0.30 0.67 Val 87 . . B B . . . −0.96 0.34 . * . −0.30 0.58 His 88 . . B B . . . −1.00 0.56 * * . −0.60 0.43 Thr 89 . . B B . . . −0.66 0.70 * . . −0.60 0.16 Phe 90 . . B B . . . −0.73 0.70 * * . −0.60 0.36 Val 91 . . B B . . . −1.08 0.63 * . . −0.26 0.14 Val 92 . . B B . . . −0.22 0.51 * * . 0.08 0.13 Asp 93 . . B B . . . −0.08 0.43 * * . 0.42 0.24 Cys 94 . . . . T T . 0.23 −0.36 * * . 2.46 0.64 Ser 95 . . . . T T . 0.93 −1.00 . * F 3.40 1.50 Asn 96 A . . . . T . 0.90 −1.64 . * F 2.66 1.50 Arg 97 A . . . . T . 1.51 −0.96 * F 2.32 1.96 Glu 98 A . . . . . . 1.21 −0.77 . * F 1.78 2.29 Asp 99 A . . . . . . 1.58 −0.77 . * F 1.44 1.91 Ile 100 A . . . . . . 1.29 −0.79 . * . 0.95 1.31 Tyr 101 A . . . . T . 1.33 −0.29 * * . 0.70 0.76 Ser 102 A . . . . T . 1.27 −0.29 * * F 0.85 0.91 Ser 103 A . . . . T . 0.41 −0.29 * . F 1.00 2.61 Ala 104 A . . . . T . 0.46 −0.33 * * F 1.00 1.23 Lys 105 A A . . . . . 0.76 −1.09 * * F 0.90 1.84 Lys 106 A A . . . . . 1.00 −0.97 * * F 0.90 1.39 Val 107 A A . . . . . 0.41 −1.36 * * F 0.90 2.38 Lys 108 A A . . . . . 0.37 −1.17 * * F 0.75 0.83 Ala 109 A A . . . . . 0.96 −0.74 * * F 0.75 0.41 Glu 110 A A . . . . . 0.06 −0.74 * * F 0.75 0.93 Ile 111 A . . B . . . −0.29 −0.74 . * F 0.75 0.34 Gly 112 A . . B . . . −0.32 −0.36 . * F 0.45 0.46 Asp 113 A . . B . . . −1.18 −0.17 . * F 0.45 0.19 Val 114 . . B B . . . −1.44 0.51 . * . −0.60 0.22 Ser 115 . . B B . . . −1.44 0.47 * . . −0.60 0.16 Ile 116 . . B B . . . −0.56 0.44 * . . −0.60 0.16 Leu 117 . . B B . . . −0.80 0.84 . * . −0.60 0.34 Val 118 . . B B . . . −1.14 0.70 . * . −0.60 0.26 Asn 119 . . B B . . . −1.14 0.74 . . −0.60 0.36 Asn 120 . . B . . T . −1.70 0.70 . . . −0.20 0.33 Ala 121 . . B . . T . −1.06 0.66 . . . −0.20 0.33 Gly 122 . . B . . T . −0.56 0.77 . . . −0.20 0.32 Val 123 . B . . T . 0.00 0.86 . . . −0.20 0.29 Val 124 . . B . . . . 0.00 0.84 . . . −0.40 0.38 Tyr 125 . . B . . . . −0.81 0.34 . . . −0.10 0.64 Thr 126 . . B . . T . −0.92 0.60 . . F −0.05 0.71 Ser 127 . . B . . T . −1.17 0.74 . . F −0.05 0.83 Asp 128 . . B . . T . −0.62 0.60 . . F −0.05 0.53 Leu 129 . . B . . T . 0.23 0.33 . . F 0.25 0.53 Phe 130 . . B . . . . 0.48 0.24 . . . −0.10 0.69 Ala 131 A . . . . . . 0.58 −0.14 . . F 0.65 0.69 Thr 132 A . . . . T . 0.88 0.29 . * F 0.40 1.29 Gln 133 A . . . . T . −0.01 0.00 . * F 0.40 2.59 Asp 134 . . . . . T C 0.80 −0.10 * . F 1.20 1.80 Pro 135 A . . . . T . 1.54 −0.60 * . F 1.30 2.16 Gln 136 A A . . . . . 1.82 −1.09 * * F 0.90 2.49 Ile 137 A A . . . . . 1.43 −1.00 * * F 0.90 2.15 Glu 138 A A . . . . . 1.43 −0.21 * * F 0.60 1.20 Lys 139 A A . . . . . 0.58 −0.64 * * F 0.90 1.20 Thr 140 A . . B . . . 0.79 −0.40 * * F 0.60 1.28 Phe 141 A . . B . . . −0.07 −0.69 * * . 0.75 1.18 Glu 142 A . . B . . . 0.01 −0.04 . * . 0.30 0.44 Val 143 A . . B . . . −0.58 0.64 . * . −0.60 0.25 Asn 144 A . . B . . . −0.66 0.66 . * . −0.60 0.29 Val 145 A . . B . . . −1.04 0.37 . * . −0.30 0.23 Leu 146 A . . B . . . −0.63 1.16 . * . −0.60 0.27 Ala 147 A . . B . . . −0.94 1.43 * * . −0.60 0.18 His 148 A . . B . . . −0.40 1.51 . * . −0.60 0.34 Phe 149 A . . B . . . −0.36 1.36 . . . −0.60 0.60 Trp 150 A . . B . . . −0.09 0.67 . . . −0.45 1.18 Thr 151 A . . B . . . 0.02 0.67 . . F −0.45 0.88 Thr 152 A . . B . . −0.20 0.96 . . F −0.45 0.88 Lys 153 A . . B . . . −0.38 0.86 . . F −0.45 0.69 Ala 154 A A . . . . . −0.27 0.37 . . . −0.30 0.74 Phe 155 A A . . . . . −0.58 0.39 . . . −0.30 0.52 Leu 156 A A . . . . . −0.58 0.51 * . . −0.60 0.26 Pro 157 A A . . . . . −0.22 1.00 * . . −0.60 0.37 Ala 158 A A . . . . . −0.27 0.50 * . . −0.60 0.84 Met 159 A A . . . . . 0.32 0.11 . . . 0.09 1.64 Thr 160 A A . . . . . 0.99 −0.17 . . F 1.08 1.71 Lys 161 A A . . . . . 1.46 −0.10 . . F 1.32 2.30 Asn 162 A . . . . T . 1.63 −0.17 . . F 1.96 2.30 Asn 163 . . . . . T C 1.33 −0.29 . . F 2.40 2.17 His 164 . . . . . T C 1.08 −0.09 . . . 1.86 0.76 Gly 165 . . . . . T C 1.08 0.56 . . . 0.72 0.35 His 166 . . B B . . . 0.18 0.64 . . . −0.12 0.32 Ile 167 . B B . . . −0.41 0.89 . . . −0.36 0.17 Val 168 . . B B . . . −0.71 0.89 . . . −0.60 0.18 Thr 169 . . B B . . . −1.27 0.84 . . . −0.60 0.17 Val 170 . . B B . . . −1.51 0.84 . . . −0.60 0.25 Ala 171 A . . B . . . −1.82 0.66 * . . −0.60 0.34 Ser 172 A . . B . . . −0.97 0.44 * . . −0.60 0.23 Ala 173 A . . . . . . −0.97 0.46 . . . −0.40 0.43 Ala 174 A . . B . . . −0.96 0.46 . * . −0.60 0.31 Gly 175 A . . B . . . −0.96 0.34 . * . −0.30 0.31 His 176 . . B B . . . −0.58 0.60 . * . −0.60 0.23 Val 177 . . B B . . . −0.98 0.53 . * . −0.60 0.35 Ser 178 . . B B . . −1.20 0.81 . * . −0.60 0.31 Val 179 . . B B . . −1.42 1.07 . * . −0.60 0.19 Pro 180 . B B . . . −1.67 1.26 . * . −0.60 0.21 Phe 181 . . B B . . . −1.88 1.11 . * . −0.60 0.16 Leu 182 . . B B . . . −1.69 1.49 . * . −0.60 0.33 Leu 183 A . . B . . . −1.69 1.41 . . . −0.60 0.11 Ala 184 A . B . . . −1.13 1.37 . . . −0.60 0.18 Tyr 185 A . . B . . . −0.88 0.97 . * . −0.60 0.29 Cys 186 A . . . . T . −0.88 0.29 . . . 0.10 0.70 Ser 187 A . . . . T . −0.66 0.39 . * . 0.10 0.60 Ser 188 A . . . . T . −0.43 0.39 . . F 0.25 0.39 Lys 189 A . . . . T . −0.70 0.13 . . F 0.25 0.73 Phe 190 A A . . . . −0.80 0.20 . . . −0.30 0.40 Ala 191 A A . . . . . −0.83 0.24 . . . −0.30 0.30 Ala 192 A A . . . . . −0.57 0.64 . . . −0.60 0.13 Val 193 A A . . . . . −0.22 1.14 * . . −0.60 0.20 Gly 194 A A . . . . . −0.58 0.36 * . . −0.30 0.40 Phe 195 A A . . . . . −0.69 0.34 . . . −0.30 0.57 His 196 A A . . . . . −0.41 0.53 * . . −0.60 0.64 Lys 197 A A . . . . . 0.18 0.37 * . F −0.15 0.93 Thr 198 A A . . . . . 1.03 −0.06 * . F 0.60 1.79 Leu 199 A A . . . . . 0.57 −0.84 * . F 0.90 2.28 Thr 200 A A . . . . . 0.68 −0.66 * . F 0.75 0.94 Asp 201 A A . . . . . 0.12 −0.16 * . F 0.45 0.66 Glu 202 A A . . . . −0.73 −0.14 * . . 0.30 0.81 Leu 203 A A . . . . . −0.42 −0.14 . . . 0.30 0.46 Ala 204 A A . . . . . −0.50 −0.23 . . . 0.30 0.48 Ala 205 A A . . . . . −0.50 0.46 . * . −0.60 0.19 Leu 206 A A . . . . . −0.84 0.94 . . . −0.60 0.34 Gln 207 A A . . . . . −1.70 0.69 . . . −0.60 0.33 Ile 208 A A . . . . . −0.84 0.83 . . . −0.60 0.24 Thr 209 . A B . . . −0.57 0.33 . . F −0.15 0.59 Gly 210 . A . . T . . −0.29 0.13 . . F 0.25 0.49 Val 211 . . B B . . . −0.14 0.21 . . F 0.00 1.01 Lys 212 . . B B . . . −0.96 0.10 . . F −0.15 0.38 Thr 213 . . B B . . . −0.73 0.30 . . F −0.15 0.31 Thr 214 . . B B . . . −0.63 0.44 . . . −0.60 0.23 Cys 215 . . B B . . . −0.29 0.23 . . . −0.30 0.17 Leu 216 . . B B . . . −0.13 0.63 . . . −0.60 0.19 Cys 217 . B . . T . −1.03 0.93 * . . −0.20 0.12 Pro 218 . . B . . T . −0.72 1.09 * . −0.20 0.16 Asn 219 . . . . T T . −0.72 0.91 * . . 0.20 0.32 Phe 220 . . B . . T . −0.40 0.71 * . . −0.20 0.85 Val 221 . . B . . . . −0.29 0.57 . . . −0.40 0.54 Asn 222 . . B . . T . −0.51 0.93 * . . −0.20 0.29 Thr 223 . . B . . T . −0.26 1.21 . . . −0.20 0.24 Gly 224 . . B . . T . −0.26 0.43 . . . −0.20 0.64 Phe 225 . . B . . T . 0.23 0.19 . . . 0.10 0.64 Ile 226 . . B . . . . 0.79 0.21 * . . −0.10 0.68 Lys 227 . . B . . . . 0.48 0.11 . . F 0.05 0.93 Asn 228 . . . . T C 0.49 0.17 * F 0.60 1.55 Pro 229 . . . . T T . 0.02 −0.23 * . F 1.40 2.95 Ser 230 . . . . T T . 0.38 −0.23 * . F 1.40 1.22 Thr 231 . . . . T T . 1.06 0.20 * . F 0.65 0.75 Ser 232 . . . . . . C 0.70 0.23 * . F 0.25 0.75 Leu 233 . . . . . . C −0.11 0.29 * * F 0.55 0.81 Gly 234 . . . . T C 0.10 0.59 . * F 0.75 0.46 Pro 235 . . . . . T C 0.19 0.10 . * F 1.35 0.60 Thr 236 . . . . . T C 0.50 0.14 . . F 1.80 1.12 Leu 237 . . . . . T C 0.80 −0.54 . . F 3.00 1.96 Glu 238 . A B . . . . 0.76 −0.97 . . F 2.10 2.19 Pro 239 A A . . . . . 0.24 −0.76 . . F 1.80 1.13 Glu 240 A A . . . . . 0.46 −0.60 * . F 1.50 1.01 Glu 241 A A . . . . . 0.88 −0.89 * * F 1.05 0.94 Val 242 A A . . . . 0.88 −0.89 * . . 0.75 1.19 Val 243 A A . . . . . 0.28 −0.63 * . . 0.60 0.57 Asn 244 A A . . . . . 0.46 −0.01 * . . 0.30 0.32 Arg 245 A A . . . . . 0.11 0.49 * . . −0.60 0.60 Leu 246 A A . . . . . −0.78 0.27 * . . −0.30 0.79 Met 247 A A . . . . . −0.73 0.31 * . . −0.30 0.35 His 248 A A . . . . . −0.19 0.60 * . . −0.60 0.15 Gly 249 A A . . . . . −0.19 1.09 * . . −0.60 0.25 Ile 250 A A . . . . . −0.30 0.40 * . . −0.60 0.45 Leu 251 A A . . . . . 0.56 0.19 * . . −0.30 0.57 Thr 252 A A . . . . . 0.56 −0.31 * . F 0.60 1.15 Glu 253 A A . . . . . −0.30 −0.13 * . F 0.60 1.62 Gln 254 A A . . . . . −0.66 −0.13 . . F 0.60 1.38 Lys 255 A A . . . . . −0.66 −0.03 . . F 0.45 0.83 Met 256 . A B . . . . −0.06 0.17 . . . −0.30 0.33 Ile 257 . A B . . . . −0.04 0.60 . . −0.60 0.30 Phe 258 . A B . . . . −0.34 0.59 . . . −0.60 0.20 Ile 259 . . B . . T . −1.23 0.97 * . . −0.20 0.27 Pro 260 . . B . . T . −1.87 1.04 . . F −0.05 0.27 Ser 261 . . . . . T C −1.97 0.86 . . F 0.15 0.32 Ser 262 . . . . . T C −1.89 0.86 . . F 0.15 0.39 Ile 263 A . . B . . . −1.50 0.86 . . . −0.60 0.21 Ala 264 A . . B . . . −0.92 0.91 * . . −0.60 0.22 Phe 265 A . . B . . . −1.52 1.01 . . . −0.60 0.24 Leu 266 A . . B . . . −1.22 1.31 * . . −0.60 0.28 Thr 267 A . . B . . . −0.81 0.63 * * . −0.60 0.49 Thr 268 A . . B . . . −0.81 0.13 * . F 0.00 1.10 Leu 269 A . . B . . . −1.03 0.03 * . F −0.15 0.94 Glu 270 A . . B . . . −0.54 0.03 * . F −0.15 0.53 Arg 271 A . . B . . . 0.27 −0.03 * . . 0.30 0.57 Ile 272 A . B . . . 0.69 −0.51 * . . 0.75 1.20 Leu 273 A . . B . . . 0.30 −1.20 * . F 0.90 1.36 Pro 274 A . . B . . . 0.30 −0.41 * . F 0.45 0.60 Glu 275 A A . . . . . −0.29 0.27 * . F −0.15 0.71 Arg 276 A A . B . . . −1.26 0.09 * . . −0.30 0.87 Phe 277 A A . B . . . −1.18 0.04 * * . −0.30 0.42 Leu 278 A A . B . . . −0.32 0.30 * * . −0.30 0.20 Ala 279 A A . B . . . 0.00 0.30 * . . −0.30 0.20 Val 280 A A . B . . . 0.04 0.30 * * . −0.30 0.46 Leu 281 A A . B . . . −0.96 −0.49 * * . 0.45 1.11 Lys 282 A A . B . . . −0.56 −0.49 * * F 0.45 0.77 Arg 283 A A . B . . . −0.60 −0.60 . * F 0.90 1.39 Lys 284 A A . B . . . 0.03 −0.60 . * F 0.90 1.25 Ile 285 A A . B . . . 0.19 −1.29 * * F 0.90 1.25 Ser 286 . A B B . . . 1.00 −0.50 * * . 0.30 0.55 Val 287 . A B B . . . 0.37 −0.50 * * . 0.30 0.46 Lys 288 . A B B . . . −0.60 0.00 * * . −0.30 0.67 Phe 289 . A B B . . . −1.53 −0.04 * * . 0.30 0.37 Asp 290 A A . B . . . −0.99 0.26 . * . −0.30 0.35 Ala 291 A A . B . . . −0.93 0.04 . * . −0.30 0.17 Val 292 A A . B . . . −0.03 0.80 . * . −0.60 0.31 Ile 293 A . . B . . . −0.68 0.01 * * . −0.30 0.37 Gly 294 A . . . . . . 0.07 0.63 . * . −0.40 0.37 Tyr 295 A A . . . . . −0.52 0.13 . * . −0.30 0.99 Lys 296 A A . . . . . 0.07 −0.01 . * . 0.45 1.42 Met 297 A A . . . . . 0.53 −0.30 . * F 0.60 2.49 Lys 298 A A . . . . . 1.03 −0.30 . * . 0.45 2.03 Ala 299 A A . . . . . 0.99 −0.63 . * . 0.75 1.30 Gln 300 A A . . . . . 0.84 −0.20 . * . 0.45 1.68

[1687]

0 SEQUENCE LISTING The patent application contains a lengthy “Sequence Listing” section. A copy of the “Sequence Listing” is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/sequence.html?DocID=20040010132). An electronic copy of the “Sequence Listing” will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3). 

What is claimed is:
 1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: (a) a polynucleotide fragment of SEQ ID NO: X or a polynucleotide fragment of the cDNA sequence included in ATCC Deposit No: Z, which is hybridizable to SEQ ID NO: X; (b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO: Y or a polypeptide fragment encoded by the cDNA sequence included in ATCC Deposit No: Z, which is hybridizable to SEQ ID NO: X; (c) a polynucleotide encoding a polypeptide domain of SEQ ID NO: Y or a polypeptide domain encoded by the cDNA sequence included in ATCC Deposit No: Z, which is hybridizable to SEQ ID NO: X; (d) a polynucleotide encoding a polypeptide epitope of SEQ ID NO: Y or a polypeptide epitope encoded by the cDNA sequence included in ATCC Deposit No: Z, which is hybridizable to SEQ ID NO: X; (e) a polynucleotide encoding a polypeptide of SEQ ID NO: Y or the cDNA sequence included in ATCC Deposit No: Z, which is hybridizable to SEQ ID NO: X, having biological activity; (f) a polynucleotide which is a variant of SEQ ID NO: X; (g) a polynucleotide which is an allelic variant of SEQ ID NO: X; (h) a polynucleotide which encodes a species homologue of the SEQ ID NO: Y; (i) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h), wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues:
 2. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a secreted protein.
 3. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO: Y or the polypeptide encoded by the cDNA sequence included in ATCC Deposit No: Z, which is hybridizable to SEQ ID NO: X.
 4. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO: X or the cDNA sequence included in ATCC Deposit No: Z, which is hybridizable to SEQ ID NO: X.
 5. The isolated nucleic acid molecule of claim 2, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.
 6. The isolated nucleic acid molecule of claim 3, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.
 7. A recombinant vector comprising the isolated nucleic acid molecule of claim
 1. 8. A method of making a recombinant host cell comprising the isolated nucleic acid molecule of claim
 1. 9. A recombinant host c l produced by the method of claim
 8. 10. The recombinant host cell of claim 9 comprising vector sequences.
 11. An isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: (a) a polypeptide fragment of SEQ ID NO: Y or the encoded sequence included in ATCC Deposit No: Z; (b) a polypeptide fragment of SEQ ID NO: Y or the encoded sequence included in ATCC Deposit No: Z, having biological activity; (c) a polypeptide domain of SEQ ID NO: Y or the encoded sequence included in ATCC Deposit No: Z; (d) a polypeptide epitope of SEQ ID NO: Y or the encoded sequence included in ATCC Deposit No: Z; (e) a secreted form of SEQ ID NO: Y or the encoded sequence included in ATCC Deposit No: Z; (f) a full length protein of SEQ ID NO: Y or the encoded sequence included in ATCC Deposit No: Z; (g) a variant of SEQ ID NO: Y; (h) an allelic variant of SEQ ID NO: Y; or (i) a species homologue of the SEQ ID NO: Y.
 12. The isolated polypeptide of claim 11, wherein the secreted form or the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.
 13. An isolated antibody that binds specifically to the isolated polypeptide of claim
 11. 14. A recombinant host cell that expresses the isolated polypeptide of claim
 11. 15. A method of making an isolated polypeptide comprising: (a) culturing the recombinant host cell of claim 14 under conditions such that said polypeptide is expressed; and (b) recovering said polypeptide.
 16. The polypeptide produced by claim
 15. 17. A method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim
 11. 18. A method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polynucleotide of claim
 1. 19. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising: (a) determining the presence or absence of a mutation in the polynucleotide of claim 1; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.
 20. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising: (a) determining the presence or amount of expression of the polypeptide of claim 11 in a biological sample; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.
 21. A method for identifying a binding partner to the polypeptide of claim 11 comprising: (a) contacting the polypeptide of claim 11 with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide.
 22. The gene corresponding to the cDNA sequence of SEQ ID NO: X.
 23. A method of identifying an activity in a biological assay, wherein the method comprises: (a) expressing SEQ ID NO: X in a cell; (b) isolating the supernatant; (c) detecting an activity in a biological assay; and (d) identifying the protein in the supernatant having the activity.
 24. The product produced by the method of claim
 20. 